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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Functional Properties of a Newly Identified C-terminal Splice Variant
of Ca<sub>v</sub>1.3 L-type Ca<sup>2+</sup> Channels<xref ref-type="fn" rid="FN1">*</xref><xref ref-type="fn" rid="FN2"><sup><inline-graphic xlink:href="sbox.jpg"></inline-graphic></sup></xref></title><author><name sortKey="Bock, Gabriella" sort="Bock, Gabriella" uniqKey="Bock G" first="Gabriella" last="Bock">Gabriella Bock</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Gebhart, Mathias" sort="Gebhart, Mathias" uniqKey="Gebhart M" first="Mathias" last="Gebhart">Mathias Gebhart</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Scharinger, Anja" sort="Scharinger, Anja" uniqKey="Scharinger A" first="Anja" last="Scharinger">Anja Scharinger</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Jangsangthong, Wanchana" sort="Jangsangthong, Wanchana" uniqKey="Jangsangthong W" first="Wanchana" last="Jangsangthong">Wanchana Jangsangthong</name><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation></author><author><name sortKey="Busquet, Perrine" sort="Busquet, Perrine" uniqKey="Busquet P" first="Perrine" last="Busquet">Perrine Busquet</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Poggiani, Chiara" sort="Poggiani, Chiara" uniqKey="Poggiani C" first="Chiara" last="Poggiani">Chiara Poggiani</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sartori, Simone" sort="Sartori, Simone" uniqKey="Sartori S" first="Simone" last="Sartori">Simone Sartori</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Mangoni, Matteo E" sort="Mangoni, Matteo E" uniqKey="Mangoni M" first="Matteo E." last="Mangoni">Matteo E. Mangoni</name><affiliation><nlm:aff id="aff3"></nlm:aff></affiliation><affiliation><nlm:aff id="aff4">INSERM, U661, F-34000 Montpellier, France,</nlm:aff></affiliation><affiliation><nlm:aff id="aff5"></nlm:aff></affiliation><affiliation><nlm:aff id="aff6">INSERM, U637, Montpellier, France</nlm:aff></affiliation></author><author><name sortKey="Sinnegger Brauns, Martina J" sort="Sinnegger Brauns, Martina J" uniqKey="Sinnegger Brauns M" first="Martina J." last="Sinnegger-Brauns">Martina J. Sinnegger-Brauns</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Herzig, Stefan" sort="Herzig, Stefan" uniqKey="Herzig S" first="Stefan" last="Herzig">Stefan Herzig</name><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation></author><author><name sortKey="Striessnig, Jorg" sort="Striessnig, Jorg" uniqKey="Striessnig J" first="Jörg" last="Striessnig">Jörg Striessnig</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Koschak, Alexandra" sort="Koschak, Alexandra" uniqKey="Koschak A" first="Alexandra" last="Koschak">Alexandra Koschak</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">21998310</idno><idno type="pmc">3234942</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234942</idno><idno type="RBID">PMC:3234942</idno><idno type="doi">10.1074/jbc.M111.269951</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">000764</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000764</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Functional Properties of a Newly Identified C-terminal Splice Variant
of Ca<sub>v</sub>1.3 L-type Ca<sup>2+</sup> Channels<xref ref-type="fn" rid="FN1">*</xref><xref ref-type="fn" rid="FN2"><sup><inline-graphic xlink:href="sbox.jpg"></inline-graphic></sup></xref></title><author><name sortKey="Bock, Gabriella" sort="Bock, Gabriella" uniqKey="Bock G" first="Gabriella" last="Bock">Gabriella Bock</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Gebhart, Mathias" sort="Gebhart, Mathias" uniqKey="Gebhart M" first="Mathias" last="Gebhart">Mathias Gebhart</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Scharinger, Anja" sort="Scharinger, Anja" uniqKey="Scharinger A" first="Anja" last="Scharinger">Anja Scharinger</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Jangsangthong, Wanchana" sort="Jangsangthong, Wanchana" uniqKey="Jangsangthong W" first="Wanchana" last="Jangsangthong">Wanchana Jangsangthong</name><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation></author><author><name sortKey="Busquet, Perrine" sort="Busquet, Perrine" uniqKey="Busquet P" first="Perrine" last="Busquet">Perrine Busquet</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Poggiani, Chiara" sort="Poggiani, Chiara" uniqKey="Poggiani C" first="Chiara" last="Poggiani">Chiara Poggiani</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sartori, Simone" sort="Sartori, Simone" uniqKey="Sartori S" first="Simone" last="Sartori">Simone Sartori</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Mangoni, Matteo E" sort="Mangoni, Matteo E" uniqKey="Mangoni M" first="Matteo E." last="Mangoni">Matteo E. Mangoni</name><affiliation><nlm:aff id="aff3"></nlm:aff></affiliation><affiliation><nlm:aff id="aff4">INSERM, U661, F-34000 Montpellier, France,</nlm:aff></affiliation><affiliation><nlm:aff id="aff5"></nlm:aff></affiliation><affiliation><nlm:aff id="aff6">INSERM, U637, Montpellier, France</nlm:aff></affiliation></author><author><name sortKey="Sinnegger Brauns, Martina J" sort="Sinnegger Brauns, Martina J" uniqKey="Sinnegger Brauns M" first="Martina J." last="Sinnegger-Brauns">Martina J. Sinnegger-Brauns</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Herzig, Stefan" sort="Herzig, Stefan" uniqKey="Herzig S" first="Stefan" last="Herzig">Stefan Herzig</name><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation></author><author><name sortKey="Striessnig, Jorg" sort="Striessnig, Jorg" uniqKey="Striessnig J" first="Jörg" last="Striessnig">Jörg Striessnig</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Koschak, Alexandra" sort="Koschak, Alexandra" uniqKey="Koschak A" first="Alexandra" last="Koschak">Alexandra Koschak</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Biological Chemistry</title><idno type="ISSN">0021-9258</idno><idno type="eISSN">1083-351X</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>An intramolecular interaction between a distal (DCRD) and a proximal regulatory
domain (PCRD) within the C terminus of long Ca<sub>v</sub>1.3 L-type
Ca<sup>2+</sup> channels (Ca<sub>v</sub>1.3<sub>L</sub>) is a major
determinant of their voltage- and Ca<sup>2+</sup>-dependent gating
kinetics. Removal of these regulatory domains by alternative splicing generates
Ca<sub>v</sub>1.3<sub>42A</sub> channels that activate at a more negative
voltage range and exhibit more pronounced Ca<sup>2+</sup>-dependent
inactivation. Here we describe the discovery of a novel short splice variant
(Ca<sub>v</sub>1.3<sub>43S</sub>) that is expressed at high levels in the
brain but not in the heart. It lacks the DCRD but, in contrast to
Ca<sub>v</sub>1.3<sub>42A</sub>, still contains PCRD. When expressed
together with α2δ1 and β3 subunits in tsA-201 cells,
Ca<sub>v</sub>1.3<sub>43S</sub> also activated at more negative voltages
like Ca<sub>v</sub>1.3<sub>42A</sub> but Ca<sup>2+</sup>-dependent
inactivation was less pronounced. Single channel recordings revealed much higher
channel open probabilities for both short splice variants as compared with
Ca<sub>v</sub>1.3<sub>L</sub>. The presence of the proximal C terminus in
Ca<sub>v</sub>1.3<sub>43S</sub> channels preserved their modulation by
distal C terminus-containing Ca<sub>v</sub>1.3- and Ca<sub>v</sub>1.2-derived
C-terminal peptides. Removal of the C-terminal modulation by alternative
splicing also induced a faster decay of Ca<sup>2+</sup> influx during
electrical activities mimicking trains of neuronal action potentials. Our
findings extend the spectrum of functionally diverse Ca<sub>v</sub>1.3 L-type
channels produced by tissue-specific alternative splicing. This diversity may
help to fine tune Ca<sup>2+</sup> channel signaling and, in the case of
short variants lacking a functional C-terminal modulation, prevent excessive
Ca<sup>2+</sup> accumulation during burst firing in neurons. This may
be especially important in neurons that are affected by
Ca<sup>2+</sup>-induced neurodegenerative processes.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Striessnig, J" uniqKey="Striessnig J">J. Striessnig</name></author><author><name sortKey="Koschak, A" uniqKey="Koschak A">A. Koschak</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Deisseroth, K" uniqKey="Deisseroth K">K. Deisseroth</name></author><author><name sortKey="Mermelstein, P G" uniqKey="Mermelstein P">P. G. Mermelstein</name></author><author><name sortKey="Xia, H" uniqKey="Xia H">H. Xia</name></author><author><name sortKey="Tsien, R W" uniqKey="Tsien R">R. W. Tsien</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Barbado, M" uniqKey="Barbado M">M. Barbado</name></author><author><name sortKey="Fablet, K" uniqKey="Fablet K">K. Fablet</name></author><author><name sortKey="Ronjat, M" uniqKey="Ronjat M">M. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id><journal-id journal-id-type="hwp">jbc</journal-id><journal-id journal-id-type="pmc">jbc</journal-id><journal-id journal-id-type="publisher-id">JBC</journal-id><journal-title-group><journal-title>The Journal of Biological Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0021-9258</issn><issn pub-type="epub">1083-351X</issn><publisher><publisher-name>American Society for Biochemistry and Molecular
Biology</publisher-name><publisher-loc>9650 Rockville Pike, Bethesda, MD 20814, U.S.A.</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">21998310</article-id><article-id pub-id-type="pmc">3234942</article-id><article-id pub-id-type="publisher-id">M111.269951</article-id><article-id pub-id-type="doi">10.1074/jbc.M111.269951</article-id><article-categories><subj-group subj-group-type="heading"><subject>Cell Biology</subject></subj-group></article-categories><title-group><article-title>Functional Properties of a Newly Identified C-terminal Splice Variant
of Ca<sub>v</sub>1.3 L-type Ca<sup>2+</sup> Channels<xref ref-type="fn" rid="FN1">*</xref><xref ref-type="fn" rid="FN2"><sup><inline-graphic xlink:href="sbox.jpg"></inline-graphic></sup></xref></article-title><alt-title alt-title-type="short">Role of Ca<sub>v</sub>1.3 C-terminal
Splicing</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bock</surname><given-names>Gabriella</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref><xref ref-type="author-notes" rid="FN3"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Gebhart</surname><given-names>Mathias</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref><xref ref-type="author-notes" rid="FN3"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Scharinger</surname><given-names>Anja</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Jangsangthong</surname><given-names>Wanchana</given-names></name><xref ref-type="aff" rid="aff2"><sup>§</sup></xref></contrib><contrib contrib-type="author"><name><surname>Busquet</surname><given-names>Perrine</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Poggiani</surname><given-names>Chiara</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sartori</surname><given-names>Simone</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Mangoni</surname><given-names>Matteo E.</given-names></name><xref ref-type="aff" rid="aff3"><sup>¶</sup></xref><xref ref-type="aff" rid="aff4"><sup>‖</sup></xref><xref ref-type="aff" rid="aff5">**</xref><xref ref-type="aff" rid="aff6"><sup>‡‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sinnegger-Brauns</surname><given-names>Martina J.</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Herzig</surname><given-names>Stefan</given-names></name><xref ref-type="aff" rid="aff2"><sup>§</sup></xref></contrib><contrib contrib-type="author"><name><surname>Striessnig</surname><given-names>Jörg</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref><xref ref-type="corresp" rid="cor1"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Koschak</surname><given-names>Alexandra</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref><xref ref-type="corresp" rid="cor2"><sup>3</sup></xref></contrib><aff id="aff1">From the<label>‡</label>Institute of Pharmacy, Pharmacology and Toxicology and Center of Molecular Biosciences Innsbruck, Peter-Mayr-Strasse 1/I, A-6020 Innsbruck, Austria,</aff><aff id="aff2">the<label>§</label>Department of Pharmacology and Center for Molecular Medicine, University of Cologne, Gleueler Strasse 24 and Robert-Koch-Strasse 21, D-50931 Cologne, Germany,</aff><aff id="aff3">the<label>¶</label>Département de Physiologie, CNRS, UMR-5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France,</aff><aff id="aff4"><label>‖</label>INSERM, U661, F-34000 Montpellier, France,</aff><aff id="aff5">the<label>**</label>Universités de Montpellier 1 & 2, UMR-5203, F-34000 Montpellier, France, and</aff><aff id="aff6"><label>‡‡</label>INSERM, U637, Montpellier, France</aff></contrib-group><author-notes><corresp id="cor1"><label>2</label> To whom correspondence may be addressed. Tel.:
<phone>43-512-507-5600</phone>; Fax: <fax>43-512-507-2931</fax>; E-mail:
<email>joerg.striessnig@uibk.ac.at</email>.</corresp><corresp id="cor2"><label>3</label> To whom correspondence may be addressed:
<addr-line>Center for Physiology and Pharmacology, Dept. of Neurophysiology
and -pharmacology, Währingerstrasse 13A, A-1090-Wien,
Austria.</addr-line> Tel.: <phone>43-1-4277-64150</phone>; Fax:
<fax>43-01-4277-9641</fax>; E-mail:
<email>alexandra.koschak@meduniwien.ac.at</email>.</corresp><fn fn-type="equal" id="FN3"><label>1</label><p>Both authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="ppub"><day>9</day><month>12</month><year>2011</year></pub-date><pub-date pub-type="epub"><day>13</day><month>10</month><year>2011</year></pub-date><pub-date pub-type="pmc-release"><day>13</day><month>10</month><year>2011</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>286</volume><issue>49</issue><fpage>42736</fpage><lpage>42748</lpage><history><date date-type="received"><day>9</day><month>6</month><year>2011</year></date><date date-type="rev-recd"><day>21</day><month>7</month><year>2011</year></date></history><permissions><copyright-statement>© 2011 by The American Society for Biochemistry and
Molecular Biology, Inc.</copyright-statement><copyright-year>2011</copyright-year><license license-type="open-access"><license-p><italic>Author's Choice</italic>—Final version full access.</license-p><license-p><ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/">Creative Commons
Attribution Non-Commercial License</ext-link> applies to Author Choice
Articles</license-p></license></permissions><self-uri xlink:title="pdf" xlink:type="simple" xlink:href="zbc04911042736.pdf"></self-uri><abstract><p>An intramolecular interaction between a distal (DCRD) and a proximal regulatory
domain (PCRD) within the C terminus of long Ca<sub>v</sub>1.3 L-type
Ca<sup>2+</sup> channels (Ca<sub>v</sub>1.3<sub>L</sub>) is a major
determinant of their voltage- and Ca<sup>2+</sup>-dependent gating
kinetics. Removal of these regulatory domains by alternative splicing generates
Ca<sub>v</sub>1.3<sub>42A</sub> channels that activate at a more negative
voltage range and exhibit more pronounced Ca<sup>2+</sup>-dependent
inactivation. Here we describe the discovery of a novel short splice variant
(Ca<sub>v</sub>1.3<sub>43S</sub>) that is expressed at high levels in the
brain but not in the heart. It lacks the DCRD but, in contrast to
Ca<sub>v</sub>1.3<sub>42A</sub>, still contains PCRD. When expressed
together with α2δ1 and β3 subunits in tsA-201 cells,
Ca<sub>v</sub>1.3<sub>43S</sub> also activated at more negative voltages
like Ca<sub>v</sub>1.3<sub>42A</sub> but Ca<sup>2+</sup>-dependent
inactivation was less pronounced. Single channel recordings revealed much higher
channel open probabilities for both short splice variants as compared with
Ca<sub>v</sub>1.3<sub>L</sub>. The presence of the proximal C terminus in
Ca<sub>v</sub>1.3<sub>43S</sub> channels preserved their modulation by
distal C terminus-containing Ca<sub>v</sub>1.3- and Ca<sub>v</sub>1.2-derived
C-terminal peptides. Removal of the C-terminal modulation by alternative
splicing also induced a faster decay of Ca<sup>2+</sup> influx during
electrical activities mimicking trains of neuronal action potentials. Our
findings extend the spectrum of functionally diverse Ca<sub>v</sub>1.3 L-type
channels produced by tissue-specific alternative splicing. This diversity may
help to fine tune Ca<sup>2+</sup> channel signaling and, in the case of
short variants lacking a functional C-terminal modulation, prevent excessive
Ca<sup>2+</sup> accumulation during burst firing in neurons. This may
be especially important in neurons that are affected by
Ca<sup>2+</sup>-induced neurodegenerative processes.</p></abstract><kwd-group><kwd>Alternative Splicing</kwd><kwd>Calcium Channels</kwd><kwd>Calcium Signaling</kwd><kwd>Cell Signaling</kwd><kwd>Ion Channels</kwd><kwd>Ca<sub>V</sub>1.3</kwd><kwd>L-type Calcium Channels</kwd><kwd>Alternative Splicing</kwd><kwd>Cellular Excitability</kwd><kwd>Ion Channels</kwd></kwd-group><funding-group><award-group><funding-source id="CS100">National Institutes of
Health</funding-source><award-id rid="CS100">R01HL087120-A2</award-id></award-group></funding-group></article-meta></front><body><sec><title>Introduction</title><p>In electrically excitable cells different types of voltage-gated
Ca<sup>2+</sup> channels convey activity-dependent intracellular
Ca<sup>2+</sup> signals with high spatial and temporal control. Such
Ca<sup>2+</sup> signals are adjusted to specific cellular needs through
many different mechanisms. At least 10 α1 subunit isoforms form the
Ca<sup>2+</sup>-selective pore of voltage-gated Ca<sup>2+</sup>channels providing diversity through differences in their biophysical properties,
the distribution within specialized plasmalemmal compartments, protein interaction
partners, and regulation by second messenger pathways (<xref ref-type="bibr" rid="B1">1</xref>). Thereby voltage-gated Ca<sup>2+</sup> channels divert
Ca<sup>2+</sup> signals to different cellular processes within the same
cell. For instance, presynaptic Ca<sub>v</sub>2.1, Ca<sub>v</sub>2.2, and
Ca<sub>v</sub>2.3 trigger fast neurotransmitter release in neurons, whereas
somatodendritic L-type Ca<sup>2+</sup> channels (LTCCs)<xref ref-type="fn" rid="FN4"><sup>4</sup></xref> (Ca<sub>v</sub>1.2, Ca<sub>v</sub>1.3) can signal
to transcriptional events and induce long lasting alterations of neuronal
responsiveness (<xref ref-type="bibr" rid="B2">2</xref>, <xref ref-type="bibr" rid="B3">3</xref>). Likewise, in mouse pancreatic β-cells the fast phase
insulin release is under the control of Ca<sub>v</sub>1.2 channels, whereas the slow
phase requires Ca<sup>2+</sup> influx through Ca<sub>v</sub>2.3 channels
(<xref ref-type="bibr" rid="B1">1</xref>, <xref ref-type="bibr" rid="B4">4</xref>).</p><p>More recently, alternative splicing has been identified as another important
regulator of voltage-gated Ca<sup>2+</sup> channel-mediated signaling.
Alternative splicing in the proximal C terminus of the Ca<sub>v</sub>2.2 α1
subunit of N-type channels changes the dynamics of G<sub>i/o</sub> protein-dependent
inhibition resulting in an altered analgetic response to spinal morphine (<xref ref-type="bibr" rid="B5">5</xref>). Alternative splicing of Ca<sub>v</sub>1.2
α1 subunits can generate variants with more hyperpolarized window currents
likely to support basal myogenic tone in arterial vascular smooth muscle (<xref ref-type="bibr" rid="B6">6</xref>).</p><p>We have recently discovered an automodulatory domain within the C terminus of
Ca<sub>v</sub>1.4 and Ca<sub>v</sub>1.3 L-type Ca<sup>2+</sup> channels
(<xref ref-type="bibr" rid="B7">7</xref>–<xref ref-type="bibr" rid="B9">9</xref>), which tightly controls
the gating behavior of these channels. This C-terminal modulator (CTM) consists of
two putative α-helices (<xref ref-type="bibr" rid="B8">8</xref>) in the
proximal (PCRD) and distal (DCRD) C terminus and interaction between the DCRD and
the PCRD is required for CTM function. The CTM strongly reduces calmodulin
(CaM)-dependent Ca<sup>2+</sup>-dependent inactivation (CDI) of
Ca<sub>v</sub>1.3 channels and promotes their activation at more negative
voltages (<xref ref-type="bibr" rid="B8">8</xref>). Because this negative activation
range of Ca<sub>v</sub>1.3 channels is a prerequisite for their dominant role in
sinoatrial node pacemaking, hearing, and for shaping neuronal excitability (<xref ref-type="bibr" rid="B1">1</xref>, <xref ref-type="bibr" rid="B10">10</xref>,
<xref ref-type="bibr" rid="B11">11</xref>) the CTM appears to have a special
role in adjusting Ca<sub>v</sub>1.3-mediated Ca<sup>2+</sup> entry. This
suggestion is further supported by the fact that alternative splicing of exon 42 can
give rise to a Ca<sub>v</sub>1.3 α1 subunit with a long
(Ca<sub>v</sub>1.3<sub>L</sub>, previously (see Ref. <xref ref-type="bibr" rid="B8">8</xref>) referred to as Ca<sub>v</sub>1.3<sub>42</sub>) and a short C
terminus (Ca<sub>v</sub>1.3<sub>42A</sub>). The latter retains the interaction
motifs for CaM but lacks the PCRD and DCRD (<xref ref-type="bibr" rid="B8">8</xref>). Using a quantitative PCR approach we demonstrated that
Ca<sub>v</sub>1.3<sub>42A</sub> mRNA is expressed at lower levels than
Ca<sub>v</sub>1.3<sub>L</sub> in whole brain, individual brain regions, and in
the heart. Ca<sub>v</sub>1.3<sub>42A</sub> channels expressed in tsA-201 cells
almost completely lack capacitative ON gating currents
(<italic>Q</italic><sub>ON</sub>) despite robust Ca<sup>2+</sup> inward
current (<italic>I</italic><sub>Ca</sub>) (<xref ref-type="bibr" rid="B8">8</xref>).
Although not yet substantiated by single channel recordings, this suggests a higher
channel open probability (<italic>P</italic><sub>open</sub>) or single channel
conductance. Thus more pronounced activity may allow this short variant to
contribute substantial <italic>I</italic><sub>Ca</sub> despite its expected lower
expression level.</p><p>Here we addressed several important questions regarding the potential physiological
significance of alternative splicing within the CTM for Ca<sub>v</sub>1.3 function.
We describe a novel splice variant (Ca<sub>v</sub>1.3<sub>43S</sub>) abundantly
expressed in the brain but not in heart tissue. This short splice variant lacks the
DCRD and therefore a functional CTM. The gating properties of
Ca<sub>v</sub>1.3<sub>43S</sub> differ slightly from
Ca<sub>v</sub>1.3<sub>42A</sub> due to the presence of the PCRD, which permits
modulation by C-terminal fragments derived from Ca<sub>v</sub>1.2 channels. Single
channel analysis revealed that <italic>P</italic><sub>open</sub> strongly increases
in the absence of a functional CTM but with no change of the single channel
conductance. We further demonstrate that C-terminal splicing changes
<italic>I</italic><sub>Ca</sub> during stimuli simulating short bursts of action
potentials. Taken together our data reveal functional differences between all
alternatively spliced Ca<sub>v</sub>1.3 channel variants investigated in this study
that are important for the understanding of their differential function in
physiological and probably also in disease states.</p></sec><sec sec-type="methods"><title>EXPERIMENTAL PROCEDURES</title><sec><title>RNA Preparation, Reverse Transcription, and PCR</title><p>Human brain total RNA was purchased from Clontech (Saint-Germain-en-Laye,
France). tsA-201 cells were trypsinized on the third day after transfection,
washed two times in PBS, and then processed immediately. Mouse tissue was
dissected from adult (6–12 months) male C57B/6N animals as published
(<xref ref-type="bibr" rid="B12">12</xref>, <xref ref-type="bibr" rid="B13">13</xref>), shock frozen in liquid nitrogen, and stored at −80
°C. Total RNA was purified from tissue pools (heart and brain regions) or
individual samples using the RNAqueus®-4PCR Kit and DNase I (brain
regions and tsA-201 cells; Ambion, Foster City, CA), the Qiagen RNeasy Lipid
Tissue Kit (whole brain and brain regions; Qiagen), and the Qiagen RNeasy
Fibrous Tissue Kit (whole heart and heart regions) according to the
manufacturer's protocols. The RNA concentration was measured using
Nanodrop, and RNA quality was evaluated via separation of 28 S and 18 S rRNA
bands on a denaturing agarose gel. One μg of total RNA was reverse
transcribed at 42 °C (RevertAid<sup>TM</sup> H Minus First Strand cDNA
Synthesis Kit, Fermentas, St. Leon-Roth, Germany) or 55 °C
(RevertAid<sup>TM</sup> Premium First Strand cDNA Synthesis Kit, Fermentas)
with random hexamer or oligo(dT) primers in a reaction volume of 20 μl.
The concentration of the cDNA was referred to in RNA equivalents,
<italic>i.e.</italic> 1 μl of reverse transcription reaction (cDNA)
was equal to 50 ng of RNA. To amplify fragments containing exon 43,
20–100 ng of RNA equivalent was used in qualitative splice variant
analysis with PCR, with primers specific for human (GenBank<sup>TM</sup>accession number <ext-link ext-link-type="gen" xlink:href="EU363339">EU363339</ext-link>; forward, 5′-AACCCTGTTTGCTTTGGTTC-3′;
reverse, 5′-GCAGCTTTGGACATATTGGC-3′) and mouse Ca<sub>v</sub>1.3
α1 subunit (NM_001083616.1; forward,
5′-CGAGCCAGAAGACTCCAAA-3′; reverse,
5′-CACAGCACTCCTCGCTACTG-3′). Alternative splicing of exon 41 was
detected using reverse primers specific for exon 42
(5′-TATAGCACGCCGGATTTCTG-3′) or exon 42A
(5′-CCACCTTCCGGAGGAGTG-3′). PCRs were performed using PCR Master
Mix (2 times) (Fermentas) in the presence of 5% (v/v) dimethyl sulfoxide
(for fragments containing exon 43; 95 °C for 5 min, 35 cycles of 92
°C for 30 s, 58 °C for 30 s, 72 °C for 1 min) or with
BioTherm<sup>TM</sup><italic>Taq</italic> DNA Polymerase (GeneCraft, Lüdinghausen, Germany) in
the presence of 1.5 m<sc>m</sc> MgCl<sub>2</sub> (for fragments containing exon
41; 94 °C for 3 min, 40 cycles of 94 °C for 45 s, 55 °C for
45 s, and 68 °C for 1 min, followed by an elongation step of 68 °C
for 10 min). Primers detecting GAPDH were used for positive control: mouse,
GenBank accession number <ext-link ext-link-type="gen" xlink:href="NM_008084">NM_008084</ext-link>, forward primer,
5′-ACTCCACTCACGGCAAATTC-3′, reverse primer,
5′-CACATTGGGGGTAGGAACAC-3′; human, GenBank accession number
<ext-link ext-link-type="gen" xlink:href="NM_002046">NM_002046</ext-link>, forward primer,
5′-CAATGACCCCTTCATTGACC-3′, reverse primer,
5′-GAGGCAGGGATGATGTTCTG-3′. Samples without template were used as
negative control. PCR products containing exons 43S or 43L were ligated into
pGEM-T Easy vector (Promega, Mannheim, Germany), verified by sequencing (MWG
Biotech), and subsequently also employed as positive and negative control
templates in PCR. Theoretically, 43S PCR fragments could have been derived from
cDNA that reflects unresolved secondary structures in the RNA region coding exon
43. Therefore, conditions that would relax the secondary structure (reverse
transcription at 55 °C, PCR in the presence of 5% (v/v) dimethyl
sulfoxide) should prevent amplification of 43S if it arose merely as a
structural artifact. We could, however, detect 43S under these conditions (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>b</italic>). As a further
control we also harvested RNA from tsA-201 cells that transiently expressed the
long Ca<sub>v</sub>1.3 variant (Ca<sub>v</sub>1.3<sub>L</sub>) to test if 43S
RNA can be artificially derived from a 43L template. <xref ref-type="fig" rid="F1">Fig. 1</xref><italic>b</italic> shows that neither cDNA isolated
from transfected tsA-201 cells nor cloned PCR fragments containing exon 43L gave
rise to 43S bands. Relative quantitative assessment of RNA expression was
investigated either with quantitative PCR using TaqMan Gene Expression assays
(Applied Biosystems) or with transcript scanning (<xref ref-type="bibr" rid="B14">14</xref>). For calculations of absolute copy numbers in
quantitative PCR, slope values of the assays for exon 42 (Mm00551393_m1), 42A
(<xref ref-type="bibr" rid="B15">15</xref>), 49 (Mm01209927_g1), and GAPDH
(Mm99999915_g1) were obtained from standard curve experiments with
assay-specific standard DNA fragments as published (<xref ref-type="bibr" rid="B12">12</xref>, <xref ref-type="bibr" rid="B15">15</xref>). Slope,
elevation, and intercepts of standard curves for exon 42 and exon 49 probes were
not significantly different. For normalization to GAPDH transcript abundance,
the slopes were confirmed to show no significant statistical differences in
whole brain samples (Graphpad Prism 5.0, Graphpad Software, San Diego, CA), and
an average slope value (−3.43 for ventral tegmental area, −3.50
for other tissues) was derived for normalization. All samples were measured in
triplicates. Samples without template and samples containing 20 ng of RNA served
as negative controls.</p><p>Quantification of the different splice forms Ca<sub>v</sub>1.3<sub>43S</sub> and
Ca<sub>v</sub>1.3<sub>43L</sub> by transcript scanning was based on
published methodology (<xref ref-type="bibr" rid="B16">16</xref>). Exon-spanning
primers 5′-CGAGCCAGAAGACTCCAAA-3′ (forward) and
5′-CACAGCACTCCTCGCTACTG-3′ (reverse) were used in PCR (95
°C for 5 min, 30 cycles of 92 °C for 30 s, 58 °C for 30 s,
72 °C for 1 min) with 50 ng of mouse whole brain or 100 ng of mouse whole
heart RNA equivalents as templates. PCR was performed using PCR Master Mix
(Fermentas) and produced two fragments of distinct size (∼400 and
∼550 bp). The PCR products were extracted from 1% agarose gels
using the NucleoSpin Kit (Macherey-Nagel), ligated with the pJET1.2/blunt vector
(Clone JET PCR Cloning Kit, Fermentas), and replicated in DH5α cells.
Resulting clones were analyzed with colony PCR (95 °C for 3 min, 30
cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min)
using primers flanking the multiple cloning site of the vector (forward,
5′-CGACTCACTATAGGGAGAGCGGC-3′; reverse,
5′-AAGAACATCGATTTTCCATGGCAG-3′). PCR products containing 43S or
43L were distinguished on 1.5% agarose gels and verified by sequencing of
the representative extracted fragments. The assay slightly overestimated the
presence of the short variant as determined using fixed ratios of cDNA templates
containing exon 43S (Ca<sub>v</sub>1.3<sub>43S</sub> α1) and 43L
(Ca<sub>v</sub>1.3<sub>L</sub> α1): 43S/43L ratio added, 0.10,
measured 0.14 (<italic>n</italic> = 116); ratio added 0.50, measured 0.69
(<italic>n</italic> = 172). This indicated an overestimation of
38–40% of the short form, and the data were corrected
accordingly.</p></sec><sec><title>Cloning of Expression Constructs</title><p>For the Ca<sub>v</sub>1.3<sub>43S</sub> splice variant, a 1217-bp fragment was
generated by PCR with primers 5′-TCGGCAGCATTATAGACGTG-3′,
5′-ATAGGATCCCTATGGAATTATGGTTATGATGGTTATGACACACCGAATTTCCTGTTTGAACACATCATCTTCTTCTTC-3′,
and human Ca<sub>v</sub>1.3<sub>L</sub> cDNA expression construct as template
(GenBank accession number <ext-link ext-link-type="gen" xlink:href="EU363339">EU363339</ext-link>). The PCR product was incorporated into the same
construct after the HindIII/BamHI digest. Ca<sub>v</sub>1.2 α1 cDNA
(GenBank accession number <ext-link ext-link-type="gen" xlink:href="X15539">X15539</ext-link>) was used to generate a N-terminal GFP-labeled
Ca<sub>V</sub>1.2<sub>C349</sub> C-terminal peptide
(GFP-Ca<sub>v</sub>1.2<sub>C349</sub>). A fragment flanked by artificially
introduced EcoRI and HindIII restriction sites was generated by PCR (forward
primer, 5′-AAG CTT GAG GGC CAC GGG TCC CC-3′; reverse primer,
5′-GAA TTC CTA CAG GCT GCT GAC GCC-3′) and subsequently ligated
with vector pGFP<sup>+</sup> (<xref ref-type="bibr" rid="B17">17</xref>)
to generate Ca<sub>v</sub>1.2 peptide 1821–2175 in the N-terminal fusion
with GFP cDNA. For all expression constructs, regions subjected to PCR were
confirmed by sequencing (MWG). Cloning of construct
GFP-Ca<sub>v</sub>1.3<sub>C158</sub> has been described (<xref ref-type="bibr" rid="B8">8</xref>).</p></sec><sec><title>Cell Culture and Transfection</title><p>For whole cell patch clamp recordings, tsA-201 cells (a human embryonic kidney
cell line stably expressing a SV40 temperature sensitive T antigen, ECACC,
number 96121229) were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal bovine serum (Invitrogen,
10270–106), 2 m<sc>m</sc> glutamine (Invitrogen, 25030-032), penicillin
(10 units/ml; Sigma, P-3032), and streptomycin (10 μg/ml; Sigma, S-6501)
and maintained at 37 °C in a humidified incubator with 10%
CO<sub>2</sub>. Cells were grown and split when they reached about
80% of confluence using 0.05% trypsin for cell dissociation. Cell
passage numbers did not exceed 20 passages. For whole cell patch clamp
recordings tsA-201 cells were transiently transfected using
Ca<sup>2+</sup>-phosphate as described previously (<xref ref-type="bibr" rid="B8">8</xref>) with an equimolar ratio of cDNA encoding
full-length Ca<sub>v</sub>1.3<sub>L</sub>, Ca<sub>v</sub>1.3<sub>42A</sub>, or
Ca<sub>v</sub>1.3<sub>43S</sub> α1-subunits together with auxiliary
Ca<sub>v</sub>β3 and Ca<sub>v</sub>α2δ1 subunits (<xref ref-type="bibr" rid="B8">8</xref>). Note that full-length human
Ca<sub>v</sub>1.3<sub>L</sub> channels are identical to
Ca<sub>v</sub>1.3<sub>42</sub> and Ca<sub>v</sub>1.3<sub>8A</sub> channels
(GenBank<sup>TM</sup> accession number <ext-link ext-link-type="gen" xlink:href="EU363339">EU363339</ext-link>) reported in our previous publications (<xref ref-type="bibr" rid="B8">8</xref>, <xref ref-type="bibr" rid="B18">18</xref>) but nomenclature has now been changed for clarity. To visualize
transfected cells either GFP alone or N-terminal GFP-labeled constructs were
used. Cells were then plated onto a 35-mm culture dish containing
poly-<sc>l</sc>-lysine-precoated coverslips. The cells were kept at 30
°C and 10% CO<sub>2</sub>, and subjected to electrophysiological
measurements 18 h after transfection.</p><p>For single channel recordings, HEK-293 cells were cultured in Petri dishes in
Dulbecco's modified Eagle's medium (DMEM; PAA, Pasching, Austria)
supplemented with 10% FBS (Sigma), penicillin (10 units/ml), and
streptomycin (10 μg/ml) (Sigma). Cells were routinely passaged twice a
week and incubated at 37 °C under 6% CO<sub>2</sub>. For transient
transfection, HEK-293 cells were seeded onto 60-mm polystyrene Petri dishes
(Falcon, Heidelberg, Germany) at a density of 1–2 × 10<sup>4</sup>cells/cm<sup>2</sup>. The amount of Ca<sub>v</sub>1.3<sub>L</sub>,
Ca<sub>v</sub>1.3<sub>42A</sub> or Ca<sub>v</sub>1.3<sub>43S</sub>α<sub>1</sub>, Ca<sub>v</sub>β<sub>3</sub>,
Ca<sub>v</sub>α<sub>1</sub>δ1, and GFP cDNA transfected was 2,
1, 1.5, and 0.5 μg, respectively. The cDNA mixture was delivered to
HEK-293 cells by Effectene® transfection (Qiagen) according to the
manufacturer's guidelines. 16–20 h after transfection, cells were
split and cultured in 35-mm polystyrene Petri dishes (Falcon, Heidelberg,
Germany) under the normal growth conditions.</p></sec><sec><title>Electrophysiological Recordings</title><sec><title></title><sec><title>Whole Cell Patch Clamp Recordings</title><p>Electrodes with a resistance of 2–5 megaohms were pulled from
glass capillaries (Borosilicate glass, 64–0792, Harvard
Apparatus) using a micropipette puller (Sutter Instruments) and
fire-polished with an MF-830 microforge (Narishige, Japan). Cells were
recorded in the whole cell configuration using Axopatch 200A amplifier
(Axon Instruments, Foster City, CA). Data were analyzed using pClamp 9
software. The pipette internal solution contained (in m<sc>m</sc>): 135
CsCl, 10 HEPES, 10 Cs-EGTA, 1 MgCl<sub>2</sub> adjusted to pH 7.4 with
CsOH (311 mosmol); bath solution: 15 CaCl<sub>2</sub>, 10 HEPES, 150
choline-Cl, and 1 MgCl<sub>2</sub>, adjusted to pH 7.4 with CsOH (320
mosmol). Current-voltage (<italic>I-V</italic>) relationships were
obtained by holding cells at a potential of −80 mV (holding
potential, HP) before applying 250-ms pulses to various test potentials.
<italic>I-V</italic> curves were fitted to the equation
<italic>I</italic> = <italic>G</italic><sub>max</sub>(<italic>V</italic> −
<italic>V</italic><sub>rev</sub>)/{1 +
exp[(<italic>V</italic> −
<italic>V</italic><sub>0.5act</sub>)/<italic>k</italic>]},
where <italic>V</italic><sub>rev</sub> is the extrapolated reversal
potential, <italic>V</italic> is the test potential, <italic>I</italic>is the peak current amplitude, <italic>G</italic><sub>max</sub> is the
maximum slope conductance, <italic>V</italic><sub>0.5,act</sub> is the
half-maximal activation voltage, and <italic>k</italic> is the slope
factor. Percentage of inactivation was determined at specified time
points during depolarizing pulses from a HP of −80 mV to the peak
current potential (<italic>V</italic><sub>max</sub>) of the
<italic>I-V</italic> relationship of the individual cell. The
voltage dependence of inactivation was assessed by application of a
20-ms test pulse to <italic>V</italic><sub>max</sub> before and after
holding cells at various conditioning test potentials for 5-s.
Steady-state inactivation curves were analyzed using the following
Boltzman relationship: <italic>I</italic> =
<italic>I</italic><sub>SS</sub> + (1 −
<italic>I</italic><sub>SS</sub>)/(1 + exp (<italic>V</italic>−
<italic>V</italic><sub>0.5,inact</sub>/<italic>k</italic><sub>inact</sub>)),
where <italic>I</italic> is the peak current amplitude,
<italic>I</italic><sub>ss</sub> is the noninactivating fraction,
<italic>V</italic> is the membrane potential,
<italic>V</italic><sub>0.5,inact</sub> is the half-inactivation
potential, and <italic>k</italic><sub>inact</sub> is the slope factor.
Experiments showing currents bigger than 3 nA were prospectively
excluded from analysis of activation and inactivation parameters to
guarantee high-quality voltage clamp. CDI was quantified as the current
remaining at the end of 250-ms depolarizations to different test
potentials (expressed as fraction of the peak current amplitude,
<italic>r</italic><sub>250</sub>). Parameter <italic>f</italic> was
defined as the maximal difference between
<italic>r</italic><sub>250</sub> values of the barium
(<italic>I</italic><sub>Ba</sub>) and calcium
(<italic>I</italic><sub>Ca</sub>) inward currents observed within
the investigated voltage range. ON-gating charge
(<italic>Q</italic><sub>ON</sub>) was measured by holding the cells
at −80 mV before applying a 20-ms test pulse to
<italic>V<sub>rev</sub></italic> where no net inward current was
observed. ON-gating currents were digitized at 50 kHz and filtered at 5
kHz and quantified by current integration over the first 2 ms of the
test pulse.</p><p>Action potential waveform (APW)-like stimulus trains were applied as ramp
steps (see <xref ref-type="fig" rid="F8">Fig. 8</xref>). Leak potentials
and capacitive transients were subtracted after blocking the current by
0.5 m<sc>m</sc> Cd<sup>2+</sup>, 0.2 m<sc>m</sc>La<sup>3+</sup>. APWs were applied 50 times at frequencies of
100, 25, and 5 Hz resulting in 10-, 40-, and 200-ms trains,
respectively. Ca<sup>2+</sup> influx was quantified by
integration of the first 80 (at 100 Hz), 320 (at 25 Hz), and 1600 ms (at
5 Hz) of <italic>I</italic><sub>Ca</sub> train activity. Percentage of
inactivation during train stimulation was determined by the ratio
between the first and the 8th <italic>I</italic><sub>Ca</sub> amplitude.
Recovery of inactivation was measured during a 10-ms test pulse to
<italic>V</italic><sub>max</sub> that were applied at various
durations after a 1-s voltage prepulse to
<italic>V</italic><sub>max</sub>. The time course of recovery was
fit to mono- or biexponential decay yielding time constants for the fast
(τ<sub>fast</sub>) and slow (τ<sub>slow</sub>)
component. The interpulse interval was 10-s. Linear leak and
capacitative currents were subtracted online with a P/4 protocol. All
voltages were corrected for the liquid junction potential of 8.5 (for 15
m<sc>m</sc> Ca<sup>2+</sup>) or 9 mV (for 2 m<sc>m</sc>Ca<sup>2+</sup>). Series resistance was compensated at
60–70%. All experiments were performed at room temperature
(∼25 °C).</p></sec><sec><title>Single Channel Patch Clamp Recordings</title><p>Single channel currents through Ca<sub>v</sub>1.3 channel splice variants
in GFP-positive cells were obtained 48–72 h after transfection.
Recording and analysis were performed at room temperature (19–23
°C) as reported (<xref ref-type="bibr" rid="B19">19</xref>, <xref ref-type="bibr" rid="B20">20</xref>). In brief, cells kept in 35-mm
culture dishes were washed and placed in a depolarizing bath solution
containing (in m<sc>m</sc>) 120 potassium glutamate, 25 KCl, 2
MgCl<sub>2</sub>, 10 HEPES, 2 EGTA, 1 CaCl<sub>2</sub>, 1
Na<sub>2</sub>ATP, 10 dextrose (pH 7.4 with KOH). Patch pipettes
made from borosilicate glass (1.7-mm diameter and 0.283-mm wall
thickness, Hilgenberg GmbH, Malsfeld, Germany) were pulled using a
Narishige PP-83 vertical puller and fire-polished using a Narishige
MF-83 microforge (Narishige Scientific Instrument Lab). Pipettes showed
typical resistances between 7 and 10 megaohm when filled with pipette
solution (in m<sc>m</sc>), 15 BaCl<sub>2</sub>, 105 triethanolamine-Cl,
10 HEPES (pH 7.4 with triethanolamine-OH). Single Ca<sup>2+</sup>channels were recorded in the cell-attached configuration at test
potentials ranging from −30 to 0 mV and depolarizing test pulses
of 150-ms duration at 1.67 Hz (HP −100 mV). Single channel
currents were amplified, filtered at 2 kHz, −3 decibel, 4-pole
Bessel, and sampled at 10 kHz with the Axopatch one-dimensional
amplifier using pClamp 5.5 software (Axon Instruments). Experiments were
analyzed whenever the channel activity persisted for at least 240 sweeps
for each test potential, with no stacked openings observed. The single
channel events were detected using pClamp 6.0 software (Axon
Instruments), and Origin 5 software (Microcal Software, Northampton, MA)
was employed for graphic analysis. Single channel gating analysis was
performed as described (<xref ref-type="bibr" rid="B20">20</xref>). To
determine unitary conductance of Ca<sub>v</sub>1.3 channels, single
channel amplitude was determine from all point histograms by fitting two
Gaussians. Single channel conductance was calculated as the slope of the
current-voltage relationship.</p></sec></sec></sec><sec><title>Statistics</title><p>All values are presented as mean ± S.E. for the indicated number of
experiments (<italic>n</italic>). For multiple comparisons statistical
significance was determined by one-way analysis of variance (ANOVA) followed by
Bonferroni multiple comparison or Dunnett's post hoc test. For comparisons
of two groups, data were analyzed by a Mann-Whitney test as indicated for
individual experiments. Statistical significance was set at <italic>p</italic>< 0.05.</p></sec></sec><sec><title>RESULTS</title><sec><title></title><sec><title></title><sec><title>Identification and Expression of Novel C Terminally Spliced
Ca<sub>v</sub>1.3 LTCCs</title><p>Based on our previous finding that alternative splicing within the C
terminus alters Ca<sup>2+</sup> influx through Ca<sub>v</sub>1.3
LTCCs, we investigated if further unknown C-terminal splice variants
exist. So far, exon 42 was believed to participate only in the formation
of functionally “long” Ca<sub>v</sub>1.3 variants
(<italic>i.e.</italic> containing both, the PCRD encoded by exon 42
and the DCRD encoded by exon 49; Ca<sub>v</sub>1.3<sub>L</sub>, <xref ref-type="fig" rid="F1">Fig. 1</xref><italic>a</italic>), which
implied that transcripts derived from exons 42 and 49 would occur with
similar abundances. However, this was not the case when we applied a
quantitative PCR approach with samples from different mouse brain
regions. The relative abundance of exon 42 exceeded the abundance of
exon 49 in all brain regions tested (<xref ref-type="fig" rid="F1">Fig.
1</xref><italic>b</italic>). This finding suggested the presence of
a substantial amount of transcripts containing exon 42 but not exon 49,
likely due to an additional alternative splicing event between exons 42
and 49. With PCR primers spanning exons 42 to 45 we indeed identified a
novel splice variant that resulted from the use of an alternative
3′ splice acceptor site, causing a shortening of exon 43 (thus
termed exon 43S; <xref ref-type="fig" rid="F1">Fig.
1</xref><italic>a</italic>). mRNA containing 43S was detected in
human (<xref ref-type="fig" rid="F2">Fig. 2</xref><italic>a</italic>)
and mouse brain (<xref ref-type="fig" rid="F2">Fig.
2</xref><italic>b</italic>). The alternative splicing event causes a
frameshift that produces a shorter C terminus and an additional unique
15-amino acid C-terminal sequence (<xref ref-type="fig" rid="F1">Fig.
1</xref><italic>a</italic>). 43S was abundant in different brain
areas but only very weakly expressed in the heart including the
sinoatrial node (SAN) (<xref ref-type="fig" rid="F2">Fig.
2</xref><italic>c</italic>). As outlined in detail under
“Experimental Procedures” we could rule out that exon 43S
PCR products were artificial transcripts (<xref ref-type="fig" rid="F2">Fig. 2</xref><italic>c</italic>).</p><fig id="F1" position="float"><label>FIGURE 1.</label><caption><p><bold>C-terminal regulatory domains and quantitative PCR using
TaqMan gene expression assays.</bold><italic>a</italic>, schematic presentation of alternative
splicing in the C terminus of Ca<sub>v</sub>1.3 channels
generating multiple Ca<sub>v</sub>1.3 α1 isoforms with
different C-terminal lengths. Constitutive and alternative exons
are shown as <italic>black</italic> and <italic>white
squares</italic>, respectively. The schematic protein domain
structure is always shown below. Ca<sub>v</sub>1.3<sub>L</sub>,
expression of exons 39 to 49 (with/without exon 44 (<xref ref-type="bibr" rid="B8">8</xref>)) yields the full-length
Ca<sub>v</sub>1.3 C terminus.
Ca<sub>v</sub>1.3<sub>43S</sub>, use of an alternative
3′ splice acceptor site in exon 43 yields a C-terminal
truncated Ca<sub>v</sub>1.3 splice variant that terminates in
the amino acid sequence, <italic>KQEIRGVITIITIIP.</italic>Ca<sub>v</sub>1.3<sub>Δ41–1</sub>,
Ca<sub>v</sub>1.3<sub>Δ41–1</sub> channels
arise from skipping exon 41 and combining with exon 42
(terminating with <italic>LDQVVPPAGGGIKDTA</italic>).
Ca<sub>v</sub>1.3<sub>Δ41–2</sub> combined
with exon 42A (terminating with <italic>LDQVVPPAGDA</italic>).
<italic>b,</italic> relative comparison of transcripts
containing exon 42 (42, <italic>black</italic>), exon 42A (42A,
<italic>dark gray</italic>), and exon 49 (49, <italic>light
gray</italic>) in brain and heart regions of WT c57Bl6/N
mice. Expression in percent (mean ± S.E.) relative to the
sum of 42 and 42A transcripts was detected in 5 whole brains
(<italic>WB</italic>), 20 ng (all others) of RNA equivalent
from individuals (WB) or tissue pools (all others; 10 animals)
are indicated. The number of experiments is given in
<italic>parentheses</italic>. Statistical analysis was
performed using one-way ANOVA followed by Bonferroni post hoc
testing; *, <italic>p</italic> < 0.05;
**, <italic>p</italic> < 0.01;
***, <italic>p</italic> < 0.001, 42
<italic>versus</italic> 49. <italic>VTA</italic>, ventral
tegmental area; <italic>Cx</italic>, cortex;
<italic>Ce</italic>, cerebellum; <italic>PFC</italic>,
prefrontal cortex; <italic>OLB</italic>, olfactory bulb;
<italic>HPC</italic>, hippocampus; <italic>AMY</italic>,
amygdala.</p></caption><graphic xlink:href="zbc0521189070001"></graphic></fig><fig id="F2" position="float"><label>FIGURE 2.</label><caption><p><bold>Detection by PCR of the C-terminal alternative splicing in
human and mouse Ca<sub>v</sub>1.3 transcripts.</bold><italic>a,</italic> fragments containing exon 43S (502 bp) or
43l (656 bp) generated by primers specific for exon 39 (forward)
and 43 (reverse) of human Ca<sub>v</sub>1.3.
<italic>WB</italic>, 500 ng of whole brain cDNA (positive
control); <italic>H<sub>2</sub>O</italic>, no template (negative
control). One representative of 4 experiments is shown.
<italic>b,</italic> fragments containing 43S (403 bp) or 43L
(557 bp) were generated by primers specific for exons 42
(forward) and 45 (reverse) of mouse Ca<sub>v</sub>1.3. Each
sample contained 20 ng of RNA equivalent. <italic>VTA</italic>,
ventral tegmental area; <italic>CP</italic>, caudate putamen;
<italic>AMY</italic>, amygdale; <italic>OLB</italic>,
olfactory bulb; <italic>HPC</italic>, hippocampus;
<italic>Ce</italic>, cerebellum; <italic>FC</italic>,
frontal cortex; <italic>PFC</italic>, prefrontal cortex. One
representative of 3 experiments is shown. <italic>c</italic>,
conditions are as described in <italic>b. WH</italic>, whole
heart; <italic>Vtr</italic>, ventricle; <italic>rA</italic>,
right atrium; <italic>lA</italic>, left atrium;
<italic>Eye</italic>, whole eye preparation; <italic>Mock
tsA</italic>, untransfected tsA-201 cells; <italic>tsA
Ca<sub>V</sub></italic>1.3<italic><sub>L</sub></italic>,
tsA-201 cells transiently expressing mouse
Ca<sub>v</sub>1.3<sub>L</sub>; all samples were tested for
GAPDH expression and contamination. <italic>GAP</italic>, PCR
with primers specific for human GAPDH. One representative of 3
experiments is shown. <italic>d,</italic> fragments containing
exons 41 (39–42, 331 bp; 39–42A, 488 bp),
Δ41–1 (39–42, 200 bp), or
Δ41–2 (39–42A, 357 bp) generated by primers
specific for exon 39 and exons 42 or 42A, respectively, of human
Ca<sub>v</sub>1.3. 1 μg of whole brain cDNA was used.
One representative of 6 independent experiments is shown.</p></caption><graphic xlink:href="zbc0521189070002"></graphic></fig><p>Unfortunately, no selective probes for quantitative real time PCR could
be obtained for 43S (not shown). We therefore applied transcript
scanning as an alternative method of quantification (<xref ref-type="bibr" rid="B14">14</xref>). Transcripts containing short
and long exon 43, respectively, were amplified from whole brain and
heart and the relative abundance of these splice variants was determined
by colony PCR (see “Experimental Procedures”). In whole
brain tissue, Ca<sub>v</sub>1.3<sub>43S</sub> variants represented 39
± 7% (<italic>n</italic> = 195) of the clones,
suggesting that Ca<sub>v</sub>1.3<sub>43S</sub> α1 subunits
account for a large proportion of Ca<sub>v</sub>1.3 α1 brain
transcripts.</p><p>Ca<sub>v</sub>1.3-mediated Ca<sup>2+</sup> influx has recently
been implicated in the selective vulnerability of dopaminergic SNc
neurons observed in Parkinson disease (<xref ref-type="bibr" rid="B21">21</xref>, <xref ref-type="bibr" rid="B22">22</xref>). Both short
variants, Ca<sub>v</sub>1.3<sub>43S</sub> (<xref ref-type="fig" rid="F2">Fig. 2</xref><italic>b</italic>) and
Ca<sub>v</sub>1.3<sub>42A</sub> (<xref ref-type="fig" rid="F1">Fig.
1</xref><italic>b</italic>), were expressed in the substantia nigra
(SN) and the adjacent ventral tegmental area. Interestingly, in real
time PCR experiments 42A-containing transcripts were more abundant in
the SN and ventral tegmental area than in other brain areas (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>b</italic>).</p><p>A much lower percentage of 43S was found in the heart (6 ±
1%, <italic>n</italic> = 213). These semiquantitative
estimates of the relative abundance of exon 43S as compared with 43L are
in good agreement with the relative abundance of the respective PCR
bands on agarose gels (<xref ref-type="fig" rid="F2">Fig. 2</xref>,
<italic>b</italic> and <italic>c</italic>). In SAN, the predominant
site of Ca<sub>v</sub>1.3 expression in the heart, 43S abundance was
comparable with whole heart and all other heart regions tested (<xref ref-type="fig" rid="F2">Fig. 2</xref><italic>c</italic>).</p><p>Our PCR screen also revealed splice variants in human brain lacking exon
41 with exon 40 either connected to exon 42 or 42A
(Δ41<sub>1</sub> and Δ41<sub>2</sub>, <xref ref-type="fig" rid="F1">Figs. 1</xref><italic>a</italic> and <xref ref-type="fig" rid="F2">2</xref><italic>d</italic>). Using selective
<italic>in situ</italic> hybridization probes for
Ca<sub>v</sub>1.3<sub>42A</sub> and Ca<sub>v</sub>1.3<sub>L</sub>together with a generic Ca<sub>v</sub>1.3 probe we found no major
differences in the expression pattern of these two isoforms in mouse
brain. This is illustrated for hippocampus, olfactory bulb, and
cerebellum in <ext-link ext-link-type="uri" xlink:href="http://www.jbc.org/cgi/content/full/M111.269951/DC1">supplemental Fig. S1</ext-link> but was also true for other brain
areas, such as the cerebral cortex and caudate putamen
(<italic>n</italic> = 3, not illustrated).</p></sec><sec><title>Ca<sub>v</sub>1.3<sub>43S</sub> Channels Show “Short”
Gating Properties</title><p>As illustrated in <xref ref-type="fig" rid="F1">Fig.
1</xref><italic>a</italic>, alternative splicing in exon 43 creates
a short variant that, in contrast to Ca<sub>v</sub>1.3<sub>42A</sub>,
retains the proximal part (PCRD) of the CTM. We therefore tested if this
difference distinguished the biophysical properties of
Ca<sub>v</sub>1.3<sub>43S</sub> from those of
Ca<sub>v</sub>1.3<sub>42A</sub> channels when expressed under
identical experimental conditions in tsA-201 cells. As expected, with 15
m<sc>m</sc> Ca<sup>2+</sup> (or Ba<sup>2+</sup>) as
charge carrier, Ca<sub>v</sub>1.3<sub>43S</sub> resembled a short
Ca<sub>v</sub>1.3 channel lacking a functional CTM (<xref ref-type="fig" rid="F3">Fig. 3</xref>, <xref ref-type="table" rid="T1">Table 1</xref>). <italic>I</italic><sub>Ca</sub> through
Ca<sub>v</sub>1.3<sub>43S</sub> channels activated and inactivated
at more negative voltages than Ca<sub>v</sub>1.3<sub>L</sub> (<xref ref-type="fig" rid="F3">Fig. 3</xref>, <italic>a</italic> and
<italic>b</italic>). Moreover, CDI (defined as the difference
between the extent of inactivation of <italic>I</italic><sub>Ba</sub><italic>versus I</italic><sub>Ca</sub>) was significantly more
pronounced (<xref ref-type="fig" rid="F3">Fig. 3</xref>,
<italic>c</italic> and <italic>d</italic>), whereas
voltage-dependent inactivation (defined as inactivation in equimolar
Ba<sup>2+</sup>) was slower (see
<italic>I</italic><sub>Ba</sub> after 250 ms, <italic>e.g.</italic>−11.5 mV in <xref ref-type="fig" rid="F3">Fig.
3</xref><italic>c</italic>). Maximal CDI essentially was as reported
for Ca<sub>v</sub>1.3<sub>42A</sub> (<italic>f</italic> = 0.6
with a maximum at +10 mV (<xref ref-type="bibr" rid="B8">8</xref>)). A more negative activation range for
Ca<sub>v</sub>1.3<sub>43S</sub> was also found for
<italic>I</italic><sub>Ba</sub> as charge carrier
(<italic>V</italic><sub>0.5,act</sub> (in mV):
Ca<sub>v</sub>1.3<sub>L</sub>, −13.8 ± 1.6,
<italic>n</italic> = 9; Ca<sub>v</sub>1.3<sub>43S</sub>,
−26.01 ± 2.5, <italic>n</italic> = 8,
<italic>p</italic> < 0.001, Mann-Whitney test). Because the
biophysical parameters obtained were very similar to those already
described by us for Ca<sub>v</sub>1.3<sub>42A</sub> (<xref ref-type="bibr" rid="B8">8</xref>) the presence of the PCRD
apparently did not affect the gating properties under these experimental
conditions.</p><fig id="F3" position="float"><label>FIGURE 3.</label><caption><p><bold>Biophysical properties of C-terminal Ca<sub>v</sub>1.3
α1 splice variants with 15 m<sc>m</sc>Ca<sup>2+</sup> as charge carrier.</bold><italic>a,</italic> mean normalized current-voltage
(<italic>I-V</italic>) curves recorded in tsA-201 cells
expressing Ca<sub>v</sub>1.3<sub>L</sub>(<italic>black</italic>), Ca<sub>v</sub>1.3<sub>43S</sub>(<italic>open circle</italic>), and
Ca<sub>v</sub>1.3<sub>42A</sub> (<italic>gray</italic>).
Activation parameters and statistics are given in <xref ref-type="table" rid="T1">Table 1</xref>.
<italic>b,</italic> steady-state inactivation curves for
Ca<sub>v</sub>1.3<sub>L</sub>,
Ca<sub>v</sub>1.3<sub>43S</sub>. Parameters for the
activation curve were obtained from parameters in
<italic>a</italic>. Inactivation parameters were as follows
in mV: <italic>V</italic><sub>0.5</sub>,
Ca<sub>v</sub>1.3<sub>L</sub>, −25.6 ± 0.52;
Ca<sub>v</sub>1.3<sub>43S</sub>, −30.9 ± 0.7,
<italic>p</italic> < 0.001; <italic>k</italic> (slope):
Ca<sub>v</sub>1.3<sub>L</sub>, −5.5 ±
0.26<sub>,</sub> Ca<sub>v</sub>1.3<sub>43S</sub>,
−4.2 ± 0.2, <italic>p</italic> = 0.027,
Mann-Whitney test. <italic>c</italic>, voltage dependence of CDI
for Ca<sub>v</sub>1.3<sub>L</sub> (<italic>left</italic>) and
Ca<sub>v</sub>1.3<sub>43S</sub> (<italic>right</italic>):
<italic>r</italic><sub>250</sub> corresponds to the fraction
of <italic>I</italic><sub>Ca</sub> or
<italic>I</italic><sub>Ba</sub> remaining after 250 ms;
<italic>f</italic> is the difference between
<italic>r</italic><sub>250</sub> values at 11.5 mV.
<italic>d</italic>, <italic>left</italic>, normalized peak
current traces of Ca<sub>v</sub>1.3<sub>L</sub>,
Ca<sub>v</sub>1.3<sub>43S</sub>, and
Ca<sub>v</sub>1.3<sub>42A</sub> evoked by 5-s depolarization
to <italic>V</italic><sub>max</sub>. <italic>Right</italic>,
percent <italic>I</italic><sub>Ca</sub> inactivation during
0.1-, 0.25-, 0.5-, 1-, and 5-s test pulses to
<italic>V</italic><sub>max</sub>. Color code as described in
<italic>a</italic>. Number of experiments is given in
<italic>parentheses. Error bars</italic> reflect S.E.
*, <italic>p</italic> < 0.05;
***, <italic>p</italic> < 0.001, one-way
ANOVA analysis followed by Bonferroni post test.</p></caption><graphic xlink:href="zbc0521189070003"></graphic></fig><table-wrap id="T1" position="float"><label>TABLE 1</label><caption><p><bold>Biophysical properties of Ca<sub>v</sub>1.3 α1
splice variants with 15 m<sc>m</sc> Ca<sup>2+</sup>as charge carrier</bold></p></caption><table frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="1" colspan="1">Channel</th><th align="center" rowspan="1" colspan="1"><italic>V</italic><sub>0.5,act</sub></th><th align="center" rowspan="1" colspan="1">Slope</th><th align="center" rowspan="1" colspan="1"><italic>V</italic><sub>max</sub></th><th align="center" rowspan="1" colspan="1"><italic>V</italic><sub>rev</sub></th><th align="center" rowspan="1" colspan="1">Activation threshold</th><th align="center" rowspan="1" colspan="1"><italic>n</italic></th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"></td><td align="center" colspan="5" rowspan="1"><italic>mV</italic></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Ca<sub>v</sub>1.3<sub>L</sub></bold></td><td align="left" rowspan="1" colspan="1">−2.4 ± 0.6</td><td align="left" rowspan="1" colspan="1">−8.6 + 0.2</td><td align="left" rowspan="1" colspan="1">12.6 + 0.5</td><td align="left" rowspan="1" colspan="1">71.9 ± 1.0</td><td align="left" rowspan="1" colspan="1">−32.6 ± 0.3</td><td align="left" rowspan="1" colspan="1">13</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Ca<sub>v</sub>1.3<sub>43S</sub></bold></td><td align="left" rowspan="1" colspan="1">−13.0 ± 0.7<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">−6.9 ± 0.2<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">1.7 ± 0.8<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">68.6 ± 0.7</td><td align="left" rowspan="1" colspan="1">−35.5 ± 0.6<xref ref-type="table-fn" rid="TF1-2"><italic><sup>b</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">16</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Ca<sub>v</sub>1.3<sub>42A</sub></bold></td><td align="left" rowspan="1" colspan="1">−12.1 ± 1.6<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">−7.5 ± 0.5<xref ref-type="table-fn" rid="TF1-3"><italic><sup>c</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">0.6 ± 3.5<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">75.9 ± 1.4</td><td align="left" rowspan="1" colspan="1">−32.0 ± 1.0<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">8</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Ca<sub>v</sub>1.3<sub>43S</sub>+ 1.3<sub>C158</sub></bold></td><td align="left" rowspan="1" colspan="1">−0.09 ± 1.6<xref ref-type="table-fn" rid="TF1-4"><italic><sup>d</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">−9.0 ± 0.3<xref ref-type="table-fn" rid="TF1-4"><italic><sup>d</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">14.6 ± 1.6<xref ref-type="table-fn" rid="TF1-4"><italic><sup>d</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">70.7 ± 2.4</td><td align="left" rowspan="1" colspan="1">−32.1 ± 1.1<xref ref-type="table-fn" rid="TF1-5"><italic><sup>e</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Ca<sub>v</sub>1.3<sub>43S</sub>+ 1.2<sub>C349</sub></bold></td><td align="left" rowspan="1" colspan="1">1.4 ± 1.3<xref ref-type="table-fn" rid="TF1-4"><italic><sup>d</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">−9.6 ± 0.2<xref ref-type="table-fn" rid="TF1-4"><italic><sup>d</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">16.6 ± 1.3<xref ref-type="table-fn" rid="TF1-4"><italic><sup>d</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">74.0 ± 1.4<xref ref-type="table-fn" rid="TF1-5"><italic><sup>e</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">−33.0 ± 0.9<xref ref-type="table-fn" rid="TF1-5"><italic><sup>e</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">12</td></tr></tbody></table><table-wrap-foot><fn id="TF1-1"><p><italic><sup>a</sup> p</italic> < 0.001, comparison with
Ca<sub>v</sub>1.3<sub>L</sub>. One-way ANOVA was
followed by Bonferroni post test.</p></fn><fn id="TF1-2"><p><italic><sup>b</sup> p</italic> < 0.01, comparison with
Ca<sub>v</sub>1.3<sub>L</sub>. One-way ANOVA was
followed by Bonferroni post test.</p></fn><fn id="TF1-3"><p><italic><sup>c</sup> p</italic> < 0.05,comparison with
Ca<sub>v</sub>1.3<sub>L</sub>. One-way ANOVA was
followed by Bonferroni post test.</p></fn><fn id="TF1-4"><p><italic><sup>d</sup> p</italic> < 0.001, comparison with
Ca<sub>v</sub>1.3<sub>43S</sub>. One-way ANOVA was
followed by Bonferroni post test.</p></fn><fn id="TF1-5"><p><italic><sup>e</sup> p</italic> < 0.05, comparison with
Ca<sub>v</sub>1.3<sub>43S</sub>. One-way ANOVA was
followed by Bonferroni post test.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Alternative Splicing Affects Channel Open Probability</title><p>We have previously found that the absence of a functional CTM in
Ca<sub>v</sub>1.3<sub>42A</sub> also decreased the ratio of
ON-charge movement (<italic>Q</italic><sub>ON</sub>, reflecting the
capacitative voltage sensor movements upon depolarization during channel
gating) to ionic tail current amplitude, which can both be measured
using a single pulse protocol (<xref ref-type="fig" rid="F4">Fig.
4</xref><italic>a</italic>). Whereas ionic current through
Ca<sub>v</sub>1.3<sub>L</sub> was always preceded by a measurable
gating current (<xref ref-type="fig" rid="F4">Fig.
4</xref><italic>a</italic>), the majority of cells with robust
<italic>I</italic><sub>Ca</sub> through
Ca<sub>v</sub>1.3<sub>43S</sub> (9 of 20) and
Ca<sub>v</sub>1.3<sub>42A</sub> (9 of 20) did not reveal any
detectable <italic>Q</italic><sub>ON</sub> (not illustrated). In cells
with measurable <italic>Q</italic><sub>ON</sub> (shown in <xref ref-type="fig" rid="F4">Fig. 4</xref>), mean
<italic>Q</italic><sub>ON</sub> was also significantly smaller for
both short variants as compared with Ca<sub>v</sub>1.3<sub>L</sub>(<italic>p</italic> < 0.001 Mann-Whitney test; <xref ref-type="fig" rid="F4">Fig. 4</xref>, <italic>a</italic> and
<italic>b</italic>) and this was seen over a wide range of
corresponding tail current amplitudes (<xref ref-type="fig" rid="F4">Fig. 4</xref><italic>c</italic>). This effect was also obvious from
the 5–10-fold increase of the slope of the
<italic>I</italic><sub>Ca</sub>-<italic>Q</italic><sub>ON</sub>relationship seen in short channels (<xref ref-type="fig" rid="F4">Fig.
4</xref><italic>c</italic>). These data indicate that both short
isoforms possess a higher open probability
(<italic>P</italic><sub>open</sub>) once the voltage sensors have
opened the channel, or a higher single channel conductance. However,
another interpretation is that the CTM immobilizes a component of the
gating current that is not required for pore opening (thereby decreasing
<italic>Q</italic><sub>ON</sub>). We therefore performed single
channel analysis to directly determine the effects of C-terminal
splicing on single channel behavior.</p><fig id="F4" position="float"><label>FIGURE 4.</label><caption><p><bold>Differential coupling of ON-gating current to the opening
of Ca<sub>v</sub>1.3 splice variant α1-subunits at
the reversal potential.</bold><italic>a</italic>, ON-gating currents were measured at
potentials at which no ionic inward and outward current was
observed (<italic>V</italic><sub>rev</sub>).
<italic>V</italic><sub>rev</sub> was determined individually
in each cell by 20-ms pulses to voltages between +70 and
+90 mV in 2-mV increments. Tail current was elicited
during repolarization to −50 mV as indicated in the step
protocol above. Representative currents are shown below (cells:
Ca<sub>v</sub>1.3<sub>L</sub>, 091009_62;
Ca<sub>v</sub>1.3<sub>43S</sub>, 081009_96;
Ca<sub>v</sub>1.3<sub>42A</sub>, 151009_15).
<italic>b</italic>, <italic>bar graphs</italic> show
ON-gating current (<italic>Q</italic><sub>ON</sub>) for
Ca<sub>v</sub>1.3<sub>L</sub> (<italic>black</italic>),
Ca<sub>v</sub>1.3<sub>43S</sub> (<italic>gray</italic>), and
Ca<sub>v</sub>1.3<sub>42A</sub> (<italic>white</italic>).
<italic>Error bars</italic> reflect S.E.
***, <italic>p</italic> < 0.001
(Mann-Whitney test). Number of experiments is given in
<italic>parenthesis. c,</italic> correlation of
<italic>Q</italic><sub>ON</sub> to maximal tail current
amplitude at <italic>V</italic><sub>rev</sub>. Color code
according to <italic>b</italic>. Calculated slopes were:
−0.0067 ± 0.0008 for
Ca<sub>v</sub>1.3<sub>L</sub>, −0.0403 ± 0.0005
for Ca<sub>v</sub>1.3<sub>43S</sub>, and −0.0705 ±
0.012 for Ca<sub>v</sub>1.3<sub>42A</sub>, respectively.</p></caption><graphic xlink:href="zbc0521189070004"></graphic></fig></sec><sec><title>Loss of C-terminal Modulation in Ca<sub>v</sub>1.3 LTCCs Increases
Single Channel Activity</title><p>On the single channel level, changes in channel activity can be due to
differences in the number of open channels, the single channel
conductance, or the probability of channel opening. To discriminate
between these possibilities we performed cell-attached single channel
experiments using 15 m<sc>m</sc> Ca<sup>2+</sup> as charge
carrier, as in our whole cell recordings. As apparent from the
representative experiments in <xref ref-type="fig" rid="F5">Fig.
5</xref>, <italic>P</italic><sub>open</sub> was much higher in both
short forms as compared with Ca<sub>v</sub>1.3<sub>L</sub> channels (see
also <ext-link ext-link-type="uri" xlink:href="http://www.jbc.org/cgi/content/full/M111.269951/DC1">supplemental Table S1</ext-link>). As a consequence, much larger
ensemble average current amplitudes were observed for the short forms
(<xref ref-type="fig" rid="F5">Fig. 5</xref>, <italic>a-c</italic>),
in good agreement with the higher slope of the
<italic>I</italic><sub>Ca</sub>-<italic>Q</italic><sub>ON</sub>relationship (<xref ref-type="fig" rid="F4">Fig.
4</xref><italic>c</italic>) and a trend to larger current densities
measured in our whole cell experiments (in pA/pF:
Ca<sub>v</sub>1.3<sub>L</sub>, 22.4 ± 4.5, <italic>n</italic>= 12; Ca<sub>v</sub>1.3<sub>43S</sub>, 32.1 ± 6.5,
<italic>n</italic> = 16; Ca<sub>v</sub>1.3<sub>42A</sub>,
44.6 ± 10.6, <italic>n</italic> = 9).
<italic>P</italic><sub>open</sub> of Ca<sub>v</sub>1.3<sub>43S</sub>was slightly lower than of Ca<sub>v</sub>1.3<sub>42A</sub>, and this
difference was significant at −30 and −10 mV. Except for
the most negative voltage that could be tested (−30 mV) the
fraction of active sweeps was comparable (<xref ref-type="fig" rid="F5">Fig. 5</xref><italic>e</italic>). Single channel currents mediated
by Ca<sub>v</sub>1.3<sub>43S</sub> and Ca<sub>v</sub>1.3<sub>42A</sub>α1 subunits were slightly, but not significantly lower compared
with Ca<sub>v</sub>1.3<sub>L</sub> (<xref ref-type="fig" rid="F5">Fig.
5</xref><italic>f</italic>).</p><fig id="F5" position="float"><label>FIGURE 5.</label><caption><p><bold>Single channel properties of Ca<sub>v</sub>1.3 C-terminal
α1 subunit splice variants.</bold><italic>a–c</italic>, representative single channel
recordings for Ca<sub>v</sub>1.3<sub>L</sub>,
Ca<sub>v</sub>1.3<sub>42A</sub>, and
Ca<sub>v</sub>1.3<sub>43S</sub> were obtained during step
depolarization to voltage ranging from −30 to 0 mV from
the holding potential of −100 mV with 15 m<sc>m</sc>Ba<sup>2+</sup> as the charge carrier. The depicted
single channel currents were elicited by 150-ms pulses to the
indicated potentials and applied every 600 ms. Ten
representative consecutive traces (of at least 300 recorded
traces per experiment) are depicted (cells:
Ca<sub>v</sub>1.3<sub>L</sub>, C9625;
Ca<sub>v</sub>1.3<sub>43S</sub>, J0427;
Ca<sub>v</sub>1.3<sub>42A</sub>, I9604). Below the
corresponding ensemble average current is presented.
<italic>d,</italic> open probability
(<italic>P</italic><sub>open</sub>) within active sweeps.
<italic>e</italic>, fraction of active sweeps containing at
least one channel opening. One-way ANOVA followed by
Dunnett's post test was performed among all test
potentials. Significance levels were: *,
<italic>p</italic> < 0.05; **,
<italic>p</italic> < 0.01; and ***,
<italic>p</italic> < 0.001 for comparing with
Ca<sub>v</sub>1.3<sub>L</sub>; +, <italic>p</italic>< 0.05 and **, <italic>p</italic> < 0.01 for
comparison with Ca<sub>v</sub>1.3<sub>43S</sub>. Data are
presented as mean ± S.E. <italic>f,</italic>current-voltage relationship of unitary currents of
Ca<sub>v</sub>1.3<sub>L</sub> (<italic>black</italic>,
<italic>n</italic> = 6),
Ca<sub>v</sub>1.3<sub>43S</sub> (<italic>light
gray</italic>, <italic>n</italic> = 8), and
Ca<sub>v</sub>1.3<sub>42A</sub> (<italic>gray</italic>,
<italic>n</italic> = 6). <italic>Solid line</italic>represents the best-fit curve to data obtained from all-point
histograms. Despite minor differences in unitary current level
at −10 mV, single channel conductance was similar for all
three isoforms (in pS: Ca<sub>v</sub>1.3<sub>L</sub>, 16.10
± 1.97, <italic>n</italic> = 5;
Ca<sub>v</sub>1.3<sub>43S</sub>, 15.94 ± 2.19,
<italic>n</italic> = 7; and
Ca<sub>v</sub>1.3<sub>42A</sub>, 14.86 ± 0.9,
<italic>n</italic> = 6).</p></caption><graphic xlink:href="zbc0521189070005"></graphic></fig></sec><sec><title>Distinct Gating Properties of the Two Short Splice Forms at
Physiological Ca<sup>2+</sup> Concentrations</title><p>Because Ca<sub>v</sub>1.3<sub>43S</sub>, but not
Ca<sub>v</sub>1.3<sub>42A</sub>, still contains the PCRD domain of
the CTM, which is located in close proximity to the CaM binding site, we
hypothesized that functional differences may be revealed at lower
extracellular Ca<sup>2+</sup> concentrations. We therefore also
studied the properties of all Ca<sub>v</sub>1.3 variants using
physiological (2 m<sc>m</sc>) extracellular Ca<sup>2+</sup> in
the bath solution. As expected from known surface charge screening
effects (<xref ref-type="bibr" rid="B23">23</xref>) activation of all
splice variants occurred at more negative voltages. As for higher
extracellular Ca<sup>2+</sup>, the short variants activated with
significantly more negative <italic>V</italic><sub>0.5,act</sub> values
(<xref ref-type="fig" rid="F6">Fig. 6</xref><italic>a</italic>,
<xref ref-type="table" rid="T2">Table 2</xref>). The negative shift
in the voltage dependence of inactivation of the two short variants was
smaller than with 15 m<sc>m</sc> Ca<sup>2+</sup> and the
difference to Ca<sub>v</sub>1.3<sub>L</sub> was not statistically
significant (<xref ref-type="fig" rid="F6">Fig.
6</xref><italic>b</italic>). Interestingly,
<italic>I</italic><sub>Ca</sub> through
Ca<sub>v</sub>1.3<sub>43S</sub> inactivated slower than
Ca<sub>v</sub>1.3<sub>42A</sub> during the first 250 ms (<xref ref-type="fig" rid="F6">Fig. 6</xref><italic>c</italic>). The effect
was also obvious from the voltage dependence of CDI. Remaining
<italic>I</italic><sub>Ca</sub> at −20 mV (voltage of maximal
difference of CDI) was significantly larger for
Ca<sub>v</sub>1.3<sub>43S</sub> than for
Ca<sub>v</sub>1.3<sub>42A</sub> (Ca<sub>v</sub>1.3<sub>43S</sub>,
0.30 ± 0.04; Ca<sub>v</sub>1.3<sub>42A</sub>, 0.19 ± 0.05;
<italic>p</italic> = 0.04; Mann-Whitney test) and its
<italic>f</italic> value was smaller (<xref ref-type="fig" rid="F6">Fig. 6</xref><italic>d</italic>). Our data clearly demonstrate that
CDI of Ca<sub>v</sub>1.3 channels differs depending on the concentration
of Ca<sup>2+</sup> in the bath solution and that the additional
sequence downstream of the CaM interaction IQ motif also including the
PCRD domain caused a small but detectable moderation of CDI in
Ca<sub>v</sub>1.3<sub>43S</sub> channels.</p><fig id="F6" position="float"><label>FIGURE 6.</label><caption><p><bold>Biophysical properties of C-terminal Ca<sub>v</sub>1.3
α1 splice variants with 2 m<sc>m</sc>Ca<sup>2+</sup> as charge carrier.</bold><italic>a</italic>, current activation properties shown in
representative of normalized <italic>I-V</italic> curves
recorded in tsA-201 cells expressing
Ca<sub>v</sub>1.3<sub>L</sub> (<italic>black</italic>),
Ca<sub>v</sub>1.3<sub>43S</sub> (<italic>gray</italic>), and
Ca<sub>v</sub>1.3<sub>42A</sub> (<italic>white</italic>).
Activation parameters and statistics are given in <xref ref-type="table" rid="T2">Table 2</xref>.
<italic>b</italic>, voltage dependence of inactivation elicited
after 5-s conditioning prepulses using 20-ms test pulses to
<italic>V</italic><sub>max</sub>. Inactivation parameters
are given in mV: <italic>V</italic><sub>0.5,inact</sub>:
Ca<sub>v</sub>1.3<sub>L</sub>, −43.4 ± 1.1,
Ca<sub>v</sub>1.3<sub>43S</sub>, −43.7 ± 0.4,
Ca<sub>v</sub>1.3<sub>42A</sub>, −46.4 ± 0.34;
<italic>k</italic> (slope): Ca<sub>v</sub>1.3<sub>L</sub>,
−5.1 ± 0.3<sub>,</sub>Ca<sub>v</sub>1.3<sub>43S</sub>, −4.7 ± 0.15,
Ca<sub>v</sub>1.3<sub>42A</sub>, −4.5 ± 0.3.
<italic>c,</italic> percent <italic>I</italic><sub>Ca</sub>inactivation during 0.1-, 0.25-, 0.5-, 1-, and 5-s test pulses
to <italic>V</italic><sub>max</sub>. Color code as described in
<italic>a. d,</italic> voltage dependence of CDI:
<italic>r<sub>250</sub></italic> corresponds to the
fraction of <italic>I</italic><sub>Ca</sub> or
<italic>I</italic><sub>Ba</sub> remaining after 250 ms;
<italic>f</italic> is the difference in
<italic>r</italic><sub>250</sub> of
<italic>I</italic><sub>Ba</sub> and
<italic>I</italic><sub>Ca</sub> at −19 mV. Number of
experiments are given in <italic>parentheses. Error
bars</italic> reflect S.E. *, <italic>p</italic> <
0.05; **, <italic>p</italic> < 0.01;
***, <italic>p</italic> < 0.001, one-way
ANOVA followed by Bonferroni post test.</p></caption><graphic xlink:href="zbc0521189070006"></graphic></fig><table-wrap id="T2" position="float"><label>TABLE 2</label><caption><p><bold>Biophysical properties of Ca<sub>v</sub>1.3 α1
splice variants with 2 m<sc>m</sc> Ca<sup>2+</sup> as
charge carrier</bold></p></caption><table frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="1" colspan="1">Channel</th><th align="center" rowspan="1" colspan="1"><italic>V</italic><sub>0.5,act</sub></th><th align="center" rowspan="1" colspan="1">Slope</th><th align="center" rowspan="1" colspan="1"><italic>V</italic><sub>max</sub></th><th align="center" rowspan="1" colspan="1"><italic>V</italic><sub>rev</sub></th><th align="center" rowspan="1" colspan="1">Activation threshold</th><th align="center" rowspan="1" colspan="1"><italic>n</italic></th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"></td><td align="center" colspan="5" rowspan="1"><italic>mV</italic></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Ca<sub>v</sub>1.3<sub>L</sub></bold></td><td align="char" char="±" rowspan="1" colspan="1">−20.5 ±
1.1</td><td align="char" char="±" rowspan="1" colspan="1">−8.1 ±
0.4</td><td align="char" char="±" rowspan="1" colspan="1">−5.0 ±
1.3</td><td align="char" char="±" rowspan="1" colspan="1">55.1 ± 0.9</td><td align="char" char="±" rowspan="1" colspan="1">−49.0 ±
0.7</td><td align="left" rowspan="1" colspan="1">12</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Ca<sub>v</sub>1.3<sub>43S</sub></bold></td><td align="char" char="±" rowspan="1" colspan="1">−28.6 ±
0.4<xref ref-type="table-fn" rid="TF2-1"><italic><sup>a</sup></italic></xref></td><td align="char" char="±" rowspan="1" colspan="1">−6.9 ±
0.2<italic><sup><xref ref-type="table-fn" rid="TF2-2">b</xref><xref ref-type="table-fn" rid="TF2-1">a</xref></sup></italic></td><td align="char" char="±" rowspan="1" colspan="1">−13.0 ±
0.5<xref ref-type="table-fn" rid="TF2-1"><italic><sup>a</sup></italic></xref></td><td align="char" char="±" rowspan="1" colspan="1">59.9 ± 2.1</td><td align="char" char="±" rowspan="1" colspan="1">−51.4 ±
0.6</td><td align="left" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1">Ca<sub>v</sub><bold>1.3<sub>42A</sub></bold></td><td align="char" char="±" rowspan="1" colspan="1">−29.0 ±
1.3<xref ref-type="table-fn" rid="TF2-3"><italic><sup>c</sup></italic></xref></td><td align="char" char="±" rowspan="1" colspan="1">−6.5 ±
0.3<xref ref-type="table-fn" rid="TF2-1"><italic><sup>a</sup></italic></xref></td><td align="char" char="±" rowspan="1" colspan="1">−14.0 ±
1.5<xref ref-type="table-fn" rid="TF2-3"><italic><sup>c</sup></italic></xref></td><td align="char" char="±" rowspan="1" colspan="1">57.4 ± 1.5</td><td align="char" char="±" rowspan="1" colspan="1">−50.2 ±
0.7</td><td align="left" rowspan="1" colspan="1">12</td></tr></tbody></table><table-wrap-foot><fn id="TF2-1"><p><italic><sup>a</sup> p</italic> < 0.01, comparison with
Ca<sub>v</sub>1.3<sub>L</sub>. One-way ANOVA followed by
Bonferroni post test.</p></fn><fn id="TF2-2"><p><italic><sup>b</sup> p</italic> < 0.05, comparison with
Ca<sub>v</sub>1.3<sub>L</sub>. One-way ANOVA followed by
Bonferroni post test.</p></fn><fn id="TF2-3"><p><italic><sup>c</sup> p</italic> < 0.001, comparison with
Ca<sub>v</sub>1.3<sub>L</sub>. One-way ANOVA followed by
Bonferroni post test.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Intermolecular Modulation of Ca<sub>v</sub>1.3<sub>43S</sub> Channels
by a Distal C-terminal Regulatory Domain of Ca<sub>v</sub>1.2
LTCCs</title><p>The presence of the PCRD domain in Ca<sub>v</sub>1.3<sub>43S</sub>channels predicts another unique modulatory mechanism. We previously
found that coexpression of a separate C-terminal peptide that contains
the DCRD can restore a fully functional CTM with full-length channel
properties in constructs lacking DCRD but still containing PCRD (<xref ref-type="bibr" rid="B8">8</xref>). <xref ref-type="fig" rid="F7">Fig. 7</xref> illustrates that this was also found for
Ca<sub>v</sub>1.3<sub>43S</sub> when coexpressed with GFP-labeled
peptide 1.3<sub>C158</sub> (GFP-1.3<sub>C158</sub>), which comprises the
last 158 amino acid residues of Ca<sub>v</sub>1.3<sub>L</sub> and
contains the DCRD. In the presence of GFP-1.3<sub>C158</sub>,
Ca<sub>v</sub>1.3<sub>43S</sub> channel behavior was
indistinguishable from Ca<sub>v</sub>1.3<sub>L</sub> with respect to the
voltage dependence of <italic>I</italic><sub>Ca</sub> activation (<xref ref-type="fig" rid="F7">Fig. 7</xref><italic>a</italic>) and
<italic>I</italic><sub>Ca</sub> inactivation during test pulses to
<italic>V</italic><sub>max</sub> (<italic>i.e.</italic> CDI) (<xref ref-type="fig" rid="F7">Fig. 7</xref>, <italic>b</italic> and
<italic>c</italic>). Because there is no published evidence for a
C-terminal post-translational proteolytic processing of
Ca<sub>v</sub>1.3<sub>L</sub> in the brain, cleaved
Ca<sub>v</sub>1.3 C termini may actually not exist as regulatory
peptides <italic>in vivo</italic>. Instead, C-terminal cleaved
Ca<sub>v</sub>1.2 channel products were found to serve as potent
noncovalently bound autoinhibitors of Ca<sub>v</sub>1.2 channels (<xref ref-type="bibr" rid="B24">24</xref>). They were also shown to exist
as separate signaling molecules that can shuttle between the nucleus and
cytoplasm in an activity-dependent manner (<xref ref-type="bibr" rid="B25">25</xref>). As Ca<sub>v</sub>1.2 and Ca<sub>v</sub>1.3 are
frequently expressed in the same cell <italic>in vivo</italic> (<xref ref-type="bibr" rid="B1">1</xref>), C-terminal Ca<sub>v</sub>1.2
peptides could modulate Ca<sub>v</sub>1.3<sub>43S</sub> channels by
binding to their PCRD. To test this possibility we co-expressed a
GFP-labeled peptide predicted to be derived from C-terminal cleavage of
Ca<sub>v</sub>1.2 (GFP-1.2<sub>C349</sub>) together with
Ca<sub>v</sub>1.3<sub>43S</sub>. GFP-1.2<sub>C349</sub> fully
restored both voltage-dependent activation and inactivation properties
of Ca<sub>v</sub>1.3<sub>L</sub> in Ca<sub>v</sub>1.3<sub>43S</sub>(<xref ref-type="fig" rid="F7">Fig. 7</xref>, for statistics, see
figure legend). Taken together these data suggest a distinct
physiological role of Ca<sub>v</sub>1.3<sub>43S</sub> channels and
define a novel functional class of Ca<sub>v</sub>1.3 channel
variants.</p><fig id="F7" position="float"><label>FIGURE 7.</label><caption><p><bold>Changes in Ca<sub>V</sub>1.3<sub>43S</sub> channel gating
properties in the presence of distal Ca<sub>V</sub>1.3 or
Ca<sub>V</sub>1.2 C-terminal peptides.</bold><italic>a</italic>, mean normalized <italic>I-V</italic> curves
recorded in tsA-201 cells expressing
Ca<sub>V</sub>1.3<sub>43S</sub> GFP-labeled peptides
1.3<sub>C158</sub> (<italic>gray dots</italic>) and
1.2<sub>C349</sub> (<italic>black dots</italic>);
<italic>I-V</italic> curves for
Ca<sub>V</sub>1.3<sub>43S</sub> and
Ca<sub>V</sub>1.3<sub>L</sub> are depicted as
<italic>gray</italic> and <italic>black fitted
lines</italic>, respectively. Activation parameters of
Ca<sub>V</sub>1.3<sub>43S</sub> + C-terminal peptides
were not statistically significant from
Ca<sub>V</sub>1.3<sub>L</sub>. For statistically significant
differences, see <xref ref-type="table" rid="T1">Table 1</xref>.
<italic>b</italic>, normalized peak currents evoked by 5-s
depolarizing steps to <italic>V</italic><sub>max</sub>.
<italic>c,</italic> percent <italic>I</italic><sub>Ca</sub>inactivation was calculated during 0.1–0.25-s test pulses
to <italic>V</italic><sub>max</sub>. Number of experiments is
given in <italic>parentheses. Error bars</italic> reflect S.E.
**, <italic>p</italic> < 0.01;
***, <italic>p</italic> < 0.001 compared
with Ca<sub>v</sub>1.3<sub>43S</sub>, one-way ANOVA was followed
by Bonferroni post test.</p></caption><graphic xlink:href="zbc0521189070007"></graphic></fig></sec><sec><title>Impact of C-terminal Modulation for Ca<sub>v</sub>1.3
Ca<sup>2+</sup> Currents during Action Potential
Waveforms</title><p>Although step depolarizations over several hundred milliseconds resemble
LTCC activity during cardiac action potentials or LTCC activity in
sensory cells, they fail to predict the consequences of C-terminal
splicing on Ca<sub>v</sub>1.3-mediated Ca<sup>2+</sup> entry
during trains of short APWs in neurons. We therefore elicited
Ca<sub>v</sub>1.3 channel activity by two different pulse protocols
mimicking different neuronal activity patterns at physiological
extracellular Ca<sup>2+</sup> concentrations. APWs simulating a
rapidly firing/bursting neuron were of short duration (5 ms) and were
elicited from a negative membrane potential (−70 mV, <xref ref-type="fig" rid="F8">Fig. 8</xref>, <italic>a–c</italic>).
100 Hz trains were included because brief high-frequency bursts also
occur <italic>in vivo</italic> in different types of neurons (<xref ref-type="bibr" rid="B26">26</xref>), are used experimentally to
induce L-type channel-dependent LTP (<xref ref-type="bibr" rid="B27">27</xref>), and were added for comparison to previously published
work (<xref ref-type="bibr" rid="B28">28</xref>) employing
Ca<sub>v</sub>1.3 channels. In addition, we mimicked activity
patterns of SNc neurons obtained from recordings in SNc neurons. Such
were elicited from more depolarized voltages (−50 mV, <xref ref-type="fig" rid="F8">Fig. 8</xref>, <italic>d–f</italic>)
at frequencies resembling either regular autonomous pacemaker activity
observed in slice recordings (5 Hz) or high frequency bursts observed
<italic>in vivo</italic> (<xref ref-type="bibr" rid="B29">29</xref>). Differences in the time course of Ca<sup>2+</sup>entry between splice variants already became apparent during the first
action potential of the APWs elicited from −70 mV (<xref ref-type="fig" rid="F8">Fig. 8</xref><italic>a</italic>). When
normalized to peak current amplitude (<italic>I</italic><sub>peak</sub>,
which occurred during the repolarization phase as tail current), short
splice variants exhibited a marked early component of
Ca<sup>2+</sup> entry evident as a distinct peak well before
the maximum of the action potential was reached (<xref ref-type="fig" rid="F8">Fig. 8</xref><italic>a</italic>, <italic>left</italic>).
During repetitive stimulation, peak inward currents through all three
splice variants decreased during APWs elicited from −70 mV (<xref ref-type="fig" rid="F8">Fig. 8</xref><italic>b</italic>), and this
was already apparent during the second APW (<xref ref-type="fig" rid="F8">Fig. 8</xref><italic>a</italic>, <italic>right</italic>).
During eight APWs at 100 or 25 Hz (<xref ref-type="fig" rid="F8">Fig.
8</xref><italic>b</italic>) twice as much peak current decayed for
Ca<sub>v</sub>1.3<sub>42A</sub> currents as compared with
Ca<sub>v</sub>1.3<sub>L</sub>. For Ca<sub>v</sub>1.3<sub>43S</sub>,
significantly more peak currents decayed at 100 Hz also (<xref ref-type="fig" rid="F8">Fig. 8</xref><italic>b</italic>,
<italic>left</italic>). At 25 Hz, Ca<sub>v</sub>1.3<sub>43S</sub>differed from Ca<sub>v</sub>1.3<sub>42A</sub> (<italic>p</italic> <
0.01, one-way ANOVA, Bonferroni post-test, <xref ref-type="fig" rid="F8">Fig. 8</xref><italic>b</italic>, <italic>right</italic>) because
the decay was indistinguishable from the long variant. Very similar
differences were found for normalized <italic>I</italic><sub>Ca</sub>integrated over eight APWs (<italic>I</italic><sub>Ca</sub> area under
first AP normalized to the 8th; <xref ref-type="fig" rid="F8">Fig.
8</xref><italic>c</italic>). During SNc-like APWs applied at 5 Hz
(reflecting autonomous pacemaking), decay of
<italic>I</italic><sub>peak</sub> was very small and similar for all
variants (<xref ref-type="fig" rid="F8">Fig. 8</xref>,
<italic>d</italic> and <italic>e</italic>, <italic>right</italic>).
In contrast, at 25 Hz (a frequency reached during short bursts recorded
<italic>in vivo</italic> (<xref ref-type="bibr" rid="B29">29</xref>)), the absence of a functional CTM caused a much larger
<italic>I</italic><sub>peak</sub> decay (<xref ref-type="fig" rid="F8">Fig. 8</xref><italic>e</italic>, <italic>left</italic>).
This was also reflected for <italic>I</italic><sub>Ca</sub> integrated
over eight stimuli (<xref ref-type="fig" rid="F8">Fig.
8</xref><italic>f</italic>).</p><fig id="F8" position="float"><label>FIGURE 8.</label><caption><p><bold>Activity-dependent <italic>I</italic><sub>Ca</sub> through
Ca<sub>v</sub>1.3 α1 subunit splice variants
during APW-like stimuli.</bold> All experiments shown in
<italic>a-f</italic> were recorded using 2 m<sc>m</sc>Ca<sup>2+</sup> as charge carrier.
<italic>a</italic>, <italic>top</italic>, APW voltage protocol
elicited from −70 mV HP composed of 3 voltage ramps:
−70 to +40, +40 to −80, and
−80 to −70 mV (afterhyperpolarization).
<italic>Bottom</italic>, normalized
<italic>I</italic><sub>Ca</sub> in response to first APW for
Ca<sub>v</sub>1.3<sub>L</sub>,
Ca<sub>v</sub>1.3<sub>43S</sub>, and
Ca<sub>v</sub>1.3<sub>42A</sub> (cells:
Ca<sub>v</sub>1.3<sub>L</sub>, 251010_95;
Ca<sub>v</sub>1.3<sub>43S</sub>, 221110_133;
Ca<sub>v</sub>1.3<sub>42A</sub>, 291010_78).
<italic>Right</italic>: the <italic>I</italic><sub>Ca</sub>response to eight APWs elicited at 100-Hz is shown for
representative experiments (cells:
Ca<sub>v</sub>1.3<sub>L</sub>, 251010_128;
Ca<sub>v</sub>1.3<sub>43S</sub>, 251010_5;
Ca<sub>v</sub>1.3<sub>42A</sub>, 151110_187).
<italic>b</italic>, decrease of the
<italic>I</italic><sub>peak</sub> during 100- and 25-Hz
trains expressed as the ratio of
<italic>I</italic><sub>peak</sub> after the 8th stimulus
divided by the <italic>I</italic><sub>peak</sub> of the first
APW. <italic>c</italic>, normalized integrated
<italic>I</italic><sub>Ca</sub> (AUC) throughout eight 100-
and 25-Hz trains (<italic>I</italic><sub>Ca</sub> during first
APW was normalized to 1). *, <italic>p</italic> <
0.05; **, <italic>p</italic> < 0.01;
***, <italic>p</italic> < 0.001
comparison to Ca<sub>v</sub>1.3<sub>L</sub>, + and
++ comparison between
Ca<sub>V</sub>1.3<sub>43S</sub> and
Ca<sub>V</sub>1.3<sub>42A</sub>; <italic>d</italic>,
<italic>top,</italic> voltage protocol of (SNc)-like APWs.
<italic>Bottom</italic>, <italic>I</italic><sub>Ca</sub> in
response to first APW for Ca<sub>v</sub>1.3<sub>L</sub>,
Ca<sub>v</sub>1.3<sub>43S</sub>, and
Ca<sub>v</sub>1.3<sub>42A</sub> α1 subunits;
<italic>curves</italic> reflect the mean from all
experiments each normalized to
<italic>I</italic><sub>peak</sub>. <italic>Right,
I</italic><sub>Ca</sub> response to eight APWs elicited at
25 Hz is shown for representative experiments (cells:
Ca<sub>v</sub>1.3<sub>L</sub>, 151210_43;
Ca<sub>v</sub>1.3<sub>43S</sub>, 101210_250;
Ca<sub>v</sub>1.3<sub>42A</sub>, 101210_140).
<italic>e</italic> and <italic>f</italic> are as described
in <italic>b</italic> and <italic>c</italic> for (SNc)-like
APWs. <italic>g,</italic> recovery of
<italic>I</italic><sub>Ca</sub> inactivation in 2
m<sc>m</sc> (<italic>left</italic>) and 15 m<sc>m</sc>Ca<sup>2+</sup> (<italic>right</italic>) for
Ca<sub>v</sub>1.3<sub>L</sub>,
Ca<sub>v</sub>1.3<sub>43S</sub>, and
Ca<sub>v</sub>1.3<sub>42A</sub>. Recovery curves were best
fitted with a bi- (Ca<sub>v</sub>1.3<sub>L</sub>) or
monoexponential function (short variants) yielding the following
time constants (τ, in ms): 15 m<sc>m</sc>Ca<sup>2+</sup>, Ca<sub>v</sub>1.3<sub>L</sub>,
τ<sub>fast</sub>, 47.4 ± 3.4;
τ<sub>slow</sub>, 385.5 ± 105; 78%
τ<sub>fast</sub>, Ca<sub>v</sub>1.3<sub>43S</sub>,
τ = 300 ± 14.2;
Ca<sub>v</sub>1.3<sub>42A</sub>, τ = 284
± 7.5; 2 m<sc>m</sc> Ca<sup>2+</sup>,
Ca<sub>v</sub>1.3<sub>L</sub>, τ<sub>fast</sub>, 150
± 102.5, τ<sub>slow</sub>, 336 ± 367.6,
57% τ<sub>fast</sub>,
Ca<sub>v</sub>1.3<sub>43S</sub>: τ = 448.3
± 14.2, Ca<sub>v</sub>1.3<sub>42A</sub>, τ
= 519.3 ± 8.2. Data are given as mean ±
S.E. All statistical comparisons were made using one-way ANOVA
and Bonferroni post test.</p></caption><graphic xlink:href="zbc0521189070008"></graphic></fig><p>The frequency-dependent larger current decay of
Ca<sub>v</sub>1.3<sub>43S</sub> and Ca<sub>v</sub>1.3<sub>42A</sub>channels during AP trains indicates a larger accumulation of channels in
inactivated states. More pronounced CDI can explain this finding.
However, differences in the recovery from inactivation could be another
contributing factor. As illustrated in <xref ref-type="fig" rid="F8">Fig. 8</xref><italic>g</italic>, Ca<sub>v</sub>1.3<sub>43S</sub>and Ca<sub>v</sub>1.3<sub>42A</sub> recovered more slowly than
Ca<sub>v</sub>1.3<sub>L</sub> from inactivation after a 1-s
conditional prepulse. This difference was independent of the
extracellular Ca<sup>2+</sup> concentration. For differences in
time constants see the legend to <xref ref-type="fig" rid="F8">Fig.
8</xref>.</p><p>Taken together, our data demonstrate that C-terminal alternative splicing
caused differences in the dynamics of Ca<sup>2+</sup> influx
during patterns of channel activity observed in neurons. In accordance
with the observation that Ca<sub>v</sub>1.3<sub>43S</sub> and
Ca<sub>v</sub>1.3<sub>42A</sub> are prominently expressed together
with Ca<sub>v</sub>1.3<sub>L</sub> in the SN this suggests that they may
contribute differentially to Ca<sup>2+</sup> signaling, depending
on the activity state of the cell.</p></sec></sec></sec></sec><sec><title>DISCUSSION</title><p>Our work provides important new insight into the tight control of the C-terminal
modulatory domain of Ca<sub>v</sub>1.3 channels. We discovered
Ca<sub>v</sub>1.3<sub>43S</sub> as a new, abundant Ca<sub>v</sub>1.3 variant
with preferential expression in the brain. Although Ca<sub>v</sub>1.3<sub>43S</sub>channels are devoid of a functional CTM and show short gating behavior, the fact
that these channels contain an additional sequence after the IQ CaM interaction
motif including the PCRD domain induced gating behavior significantly different from
Ca<sub>v</sub>1.3<sub>42A</sub>. We provide direct evidence in single channel
recordings that the presence of the Ca<sub>v</sub>1.3-CTM causes a strong reduction
of the <italic>P</italic><sub>open</sub> of the channel. Moreover, we demonstrate
that alternative splicing in the C terminus significantly changes the dynamics of
Ca<sub>v</sub>1.3-mediated Ca<sup>2+</sup> entry during electrical
activity mimicking different neuronal firing patterns.</p><p>Our discovery of Ca<sub>v</sub>1.3<sub>43S</sub> now defines four classes of
functionally distinct Ca<sub>v</sub>1.3 channel variants generated by alternative
splicing in the C terminus. One class of Ca<sub>v</sub>1.3 channels lacks a
functional IQ domain that is a major site for CaM interaction (<xref ref-type="bibr" rid="B30">30</xref>). This class results from a frameshift in exon 41 caused by
different acceptor site usage and is expressed in the inner ear (<xref ref-type="bibr" rid="B31">31</xref>) as well as in pancreatic islets (<xref ref-type="bibr" rid="B32">32</xref>). CaM-mediated CDI is absent in this variant
(<xref ref-type="bibr" rid="B31">31</xref>). Notably, we report here two new
splice variants expressed in human and mouse brain that also lack a functional IQ
domain but result from the omission of exon 41 and direct linking of exon 40 with
either exon 42 or 42A. We did not further characterize these transcripts in this
study. A second and third class corresponds to Ca<sub>v</sub>1.3<sub>L</sub> and
Ca<sub>v</sub>1.3<sub>42A</sub> channels that either contain or completely lack
the two CTM domains (PCRD and DCRD) (<xref ref-type="bibr" rid="B8">8</xref>).
Ca<sub>v</sub>1.3<sub>43S</sub> channels define a new fourth class, because they
still contain the PCRD domain. Despite biophysical properties very similar to
Ca<sub>v</sub>1.3<sub>42A</sub> we found several differences between these two
short forms: the <italic>P</italic><sub>open</sub> of
Ca<sub>v</sub>1.3<sub>43S</sub> differed from Ca<sub>v</sub>1.3<sub>42A</sub> at
physiologically relevant test potentials, CDI in Ca<sub>v</sub>1.3<sub>43S</sub>channels was less pronounced with 2 m<sc>m</sc> Ca<sup>2+</sup> as the charge
carrier, and compared with Ca<sub>v</sub>1.3<sub>L</sub> 25 Hz APWs from −70
mV caused a more pronounced peak current decay during brief trains in
Ca<sub>v</sub>1.3<sub>42A</sub> but not in Ca<sub>v</sub>1.3<sub>43S</sub>channels. These data indicate that either the presence of the PCRD or the additional
15-amino acid peptide forming a unique C terminus (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>a</italic>) can still exert functional effects on channel
gating. The molecular mechanism, however, still needs to be clarified. Interference
of this sequence stretch with CaM binding/effector sites that are located adjacent
to the PCRD domain is one possible explanation for the slight moderation of CDI in
Ca<sub>v</sub>1.3<sub>43S</sub> channels.</p><p>Another distinguishing feature of Ca<sub>v</sub>1.3<sub>43S</sub> is its capability
to bind peptides containing the DCRD and thereby form a fully functional CTM. We
show that this is not limited to the DCRD of Ca<sub>v</sub>1.3 channels because a
peptide with a Ca<sub>v</sub>1.2-derived DCRD also induced gating properties
indistinguishable from the long form of Ca<sub>v</sub>1.3. Although C-terminal
proteolytical processing has not been detected so far for Ca<sub>v</sub>1.3
channels, the C termini of both Ca<sub>v</sub>1.1 (skeletal muscle (<xref ref-type="bibr" rid="B33">33</xref>)) and Ca<sub>v</sub>1.2 (brain (<xref ref-type="bibr" rid="B25">25</xref>, <xref ref-type="bibr" rid="B34">34</xref>)
and heart (<xref ref-type="bibr" rid="B24">24</xref>, <xref ref-type="bibr" rid="B35">35</xref>)) are post-translationally cleaved. The Ca<sub>v</sub>1.2
fragment either remains noncovalently associated with the channel (<xref ref-type="bibr" rid="B24">24</xref>) or exists as a separate peptide that can
translocate to the nucleus from the cytoplasm in an activity-dependent manner (<xref ref-type="bibr" rid="B25">25</xref>). It is therefore possible that this peptide
also binds to Ca<sub>v</sub>1.3<sub>43S</sub> either via a potential signaling link
between Ca<sub>v</sub>1.2 and Ca<sub>v</sub>1.3 channels or even via a nuclear
signaling pathway. Our findings therefore provide a rational basis for further
biochemical experiments to prove the existence of such complexes <italic>in
vivo</italic>. However, one has to take into account that this would require
immunoprecipitation experiments with specific antibodies directed against
Ca<sub>v</sub>1.3<sub>43S</sub> employing extracts from a (limited) subset of
brain regions where the cleaved fragment is most abundant (<xref ref-type="bibr" rid="B25">25</xref>).</p><p>Our quantitative and semi-quantitative PCR data revealed that the two short variants
are primarily expressed in mouse and human brain but occur at much lower abundance
in heart. Previous work was based on the assumption that inclusion of exon 42A
results in Ca<sub>v</sub>1.3 variants with short C termini, whereas inclusion of
exon 42 invariably results in a long C terminus encoded by exons 42 to 49 (with or
without exon 44, (<xref ref-type="bibr" rid="B8">8</xref>)). Here we demonstrate
that exon 42 may also be part of the short variant Ca<sub>v</sub>1.3<sub>43S</sub>.
If exon 42 can be linked to either 43 (permitting long transcripts) or 43S, and if
there is no further truncating splicing downstream, then the sum of full-length
transcripts (reflected by the abundance of exon 49) and transcripts containing 43S
should approximately equal the number of transcripts containing exon 42. In whole
brain this appears to be the case, because we found that at least 39% of exon
43-derived transcripts comprise 43S, and exon 49 is abundantly expressed (<xref ref-type="fig" rid="F1">Fig. 1</xref>, <italic>a</italic> and
<italic>b</italic>). A larger discrepancy was found in the SAN. Less than 10%
of transcripts in SAN contained 43S, suggesting that in the heart exon 42
preferentially combines with long exon 43. Thus, there should be predominant
abundance of full-length transcripts. However, exon 49 as our full-length transcript
“indicator” was also not very abundant in the SAN, and exons 42A
+ 49 did not add up to the exon 42 expression level (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>b</italic>). This indicates that one or more yet
unidentified splice variants (presumably with short gating characteristics) might
exist in the SAN. In a numerical modeling of mouse SAN automaticity (<ext-link ext-link-type="uri" xlink:href="http://www.jbc.org/cgi/content/full/M111.269951/DC1">supplemental
Fig. S2</ext-link>) we showed that <italic>I</italic><sub>Ca</sub> mediated by
both Ca<sub>v</sub>1.3<sub>L</sub> and Ca<sub>v</sub>1.3<sub>42A</sub> can sustain
pacemaking when using the biophysical properties of the two variants recorded in 2
m<sc>m</sc> extracellular Ca<sup>2+</sup>.
Ca<sub>v</sub>1.3<sub>42A</sub>-mediated <italic>I</italic><sub>Ca</sub>generated a SAN pacing rate that was slightly faster than that of
Ca<sub>v</sub>1.3<sub>L</sub> (<ext-link ext-link-type="uri" xlink:href="http://www.jbc.org/cgi/content/full/M111.269951/DC1">supplemental
Fig. S2</ext-link>). Simulation of <italic>I</italic><sub>CaL</sub> behavior
during pacemaking showed the predicted current peak of
Ca<sub>v</sub>1.3<sub>L</sub>-mediated <italic>I</italic><sub>CaL</sub> during the
action potential plateau and the first repolarization phases were higher than that
of Ca<sub>v</sub>1.3<sub>42A</sub>-mediated <italic>I</italic><sub>CaL</sub>(<ext-link ext-link-type="uri" xlink:href="http://www.jbc.org/cgi/content/full/M111.269951/DC1">supplemental
Fig. S2<italic>b</italic></ext-link>). This prediction is consistent with the
faster inactivation kinetics of Ca<sub>v</sub>1.3<sub>42A</sub>-mediated
<italic>I</italic><sub>CaL</sub> and accounted for the slightly shorter action
potential duration of Ca<sub>v</sub>1.3<sub>L</sub>-mediated pacemaking (<ext-link ext-link-type="uri" xlink:href="http://www.jbc.org/cgi/content/full/M111.269951/DC1">supplemental
Fig. S2<italic>a</italic></ext-link>).</p><p>We also found pronounced differences for <italic>I</italic><sub>Ca</sub> mediated by
the different splice variants during neuron-like activity patterns, in particular
between long Ca<sub>v</sub>1.3<sub>L</sub> and short forms. These differences were
frequency dependent and mainly seen at higher stimulation rates. We predict that
somatodendritic expression of short variants, associated with a more pronounced
current decay during high frequency bursts (as suggested by our data), would cause
smaller Ca<sup>2+</sup> accumulation and may affect the strength and/or
dynamics of other Ca<sup>2+</sup>-dependent processes, in particular coupling
to Ca<sup>2+</sup>-activated K<sup>+</sup> channels (<xref ref-type="bibr" rid="B33">33</xref>, <xref ref-type="bibr" rid="B36">36</xref>),
excitation transcription coupling (<xref ref-type="bibr" rid="B2">2</xref>, <xref ref-type="bibr" rid="B3">3</xref>), or Ca<sup>2+</sup> release from
intracellular stores (<xref ref-type="bibr" rid="B37">37</xref>). Such mechanisms
may help a neuron to prevent an excessive increase in intracellular
Ca<sup>2+</sup> during bursting and be especially important,
<italic>e.g.</italic> for SNc neurons in which Ca<sub>v</sub>1.3 LTCCs have been
found to contribute to their selective vulnerability in Parkinson disease (<xref ref-type="bibr" rid="B21">21</xref>) and where Ca<sup>2+</sup> buffering
capacity is low (<xref ref-type="bibr" rid="B38">38</xref>). These neurons have even
been reported to switch from a low frequency rhythmic firing mode (up to around 5
Hz) to a bursting mode with 2–10 APs of higher frequency (up to 30 Hz) in
response to unexpected presentation of primary rewards (<xref ref-type="bibr" rid="B29">29</xref>, <xref ref-type="bibr" rid="B39">39</xref>). During burst
activity intracellular Ca<sup>2+</sup> is known to rise to much higher levels
(<xref ref-type="bibr" rid="B37">37</xref>). We showed in this study that both
short forms (in particular Ca<sub>v</sub>1.3<sub>42A</sub>) are expressed in the SNc
in addition to the long form. It will therefore be particularly interesting to
examine the possibility of whether the ratio of these variants is also altered under
pathophysiological conditions in the neurodegenerative process of Parkinson
disease.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material id="PMC_1" content-type="local-data"><caption><title>Supplemental Data</title></caption><media mimetype="text" mime-subtype="html" xlink:href="supp_286_49_42736__index.html"></media><media xlink:role="associated-file" mimetype="application" mime-subtype="pdf" xlink:href="supp_M111.269951_jbc.M111.269951-3.pdf"></media><media xlink:role="associated-file" mimetype="application" mime-subtype="pdf" xlink:href="supp_M111.269951_jbc.M111.269951-2.pdf"></media><media xlink:role="associated-file" mimetype="application" mime-subtype="pdf" xlink:href="supp_M111.269951_jbc.M111.269951-1.pdf"></media><media xlink:role="associated-file" mimetype="application" mime-subtype="pdf" xlink:href="supp_M111.269951_jbc.M111.269951-4.pdf"></media></supplementary-material></sec></body><back><fn-group><fn fn-type="supported-by" id="FN1"><label>*</label><p>This work was supported, in whole or in part, by National Institutes of Health
Grant R01HL087120-A2, Austrian Science Fund (FWF) Grants P-22528 (to A. K.) and
P-20670 and F4402 (to J. S.), The European Community Research Training Network
Cavnet Grant MRTN-CT-2006-35367), Center for Molecular Medicine Cologne Grant
CMMC A5 (to S. H.), and the University of Innsbruck.</p></fn><fn fn-type="supplementary-material" id="FN2"><label><inline-graphic xlink:href="sbox.jpg"></inline-graphic></label><p>The on-line version of this article (available at <ext-link ext-link-type="uri" xlink:href="http://www.jbc.org">http://www.jbc.org</ext-link>) contains <ext-link ext-link-type="uri" xlink:href="http://www.jbc.org/cgi/content/full/M111.269951/DC1">supplemental Figs. S1 and S2 and Table S1</ext-link>.</p></fn></fn-group><fn-group content-type="abbreviations"><fn id="FN4"><label>4</label><p>The abbreviations used are: <def-list><def-item><term id="G1">LTCC</term><def><p>L-type Ca<sup>2+</sup> channel</p></def></def-item><def-item><term id="G2">CDI</term><def><p>calcium-dependent inactivation</p></def></def-item><def-item><term id="G3">CaM</term><def><p>calmodulin</p></def></def-item><def-item><term id="G4">CTM</term><def><p>C-terminal modulator</p></def></def-item><def-item><term id="G5">PCRD</term><def><p>proximal C-terminal regulatory domain</p></def></def-item><def-item><term id="G6">DCRD</term><def><p>distal C-terminal regulatory domain</p></def></def-item><def-item><term id="G7">HP</term><def><p>holding potential</p></def></def-item><def-item><term id="G8">APW</term><def><p>action potential waveform</p></def></def-item><def-item><term id="G9">ANOVA</term><def><p>one-way analysis of variance</p></def></def-item><def-item><term id="G10">SAN</term><def><p>sinoatrial node</p></def></def-item><def-item><term id="G11">SN</term><def><p>substantia nigra.</p></def></def-item></def-list></p></fn></fn-group><ack><title>Acknowledgments</title><p>We thank Jenny Müller, Michaela Hatzl, and Gospava Stojanovic for excellent
technical assistance.</p></ack><notes><title>Note Added in Proof</title><p>Splice variant Cav1.343S and additional C-terminal splice variants are described in
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