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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Singular Interaction between an Antimetastatic Agent and the Lipid Bilayer:
The Ohmline Case</title>
<author><name sortKey="Herrera, Fernando X0a E" sort="Herrera, Fernando X0a E" uniqKey="Herrera F" first="Fernando X0a E." last="Herrera">Fernando X0a E. Herrera</name>
<affiliation><nlm:aff id="aff1">Physics Department,<institution>Universidad Nacional del Litoral, Ciudad Universitaria</institution>
, 3000 Santa Fe,<country>Argentina</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Sevrain, Charlotte M" sort="Sevrain, Charlotte M" uniqKey="Sevrain C" first="Charlotte M." last="Sevrain">Charlotte M. Sevrain</name>
<affiliation><nlm:aff id="aff2"><institution>Université de Brest, CEMCA, UMR CNRS 6521, IBSAM</institution>
, 6, Avenue Victor le Gorgeu, 29238 Brest,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Jaffres, Paul Alain" sort="Jaffres, Paul Alain" uniqKey="Jaffres P" first="Paul-Alain" last="Jaffrès">Paul-Alain Jaffrès</name>
<affiliation><nlm:aff id="aff2"><institution>Université de Brest, CEMCA, UMR CNRS 6521, IBSAM</institution>
, 6, Avenue Victor le Gorgeu, 29238 Brest,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Couthon, Helene" sort="Couthon, Helene" uniqKey="Couthon H" first="Hélène" last="Couthon">Hélène Couthon</name>
<affiliation><nlm:aff id="aff2"><institution>Université de Brest, CEMCA, UMR CNRS 6521, IBSAM</institution>
, 6, Avenue Victor le Gorgeu, 29238 Brest,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Grelard, Axelle" sort="Grelard, Axelle" uniqKey="Grelard A" first="Axelle" last="Grélard">Axelle Grélard</name>
<affiliation><nlm:aff id="aff4"><institution>Université Bordeaux, Institute of Chemistry & Biology of Membranes & Nanoobjects, UMR5248 CNRS</institution>
, Allée de Geoffroy St Hilaire Bât B14 Pessac, 33600 Bordeaux,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Dufourc, Erick J" sort="Dufourc, Erick J" uniqKey="Dufourc E" first="Erick J." last="Dufourc">Erick J. Dufourc</name>
<affiliation><nlm:aff id="aff4"><institution>Université Bordeaux, Institute of Chemistry & Biology of Membranes & Nanoobjects, UMR5248 CNRS</institution>
, Allée de Geoffroy St Hilaire Bât B14 Pessac, 33600 Bordeaux,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chantome, Aurelie" sort="Chantome, Aurelie" uniqKey="Chantome A" first="Aurélie" last="Chantôme">Aurélie Chantôme</name>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Potier Cartereau, Marie" sort="Potier Cartereau, Marie" uniqKey="Potier Cartereau M" first="Marie" last="Potier-Cartereau">Marie Potier-Cartereau</name>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Vandier, Christophe" sort="Vandier, Christophe" uniqKey="Vandier C" first="Christophe" last="Vandier">Christophe Vandier</name>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Bouchet, Ana M" sort="Bouchet, Ana M" uniqKey="Bouchet A" first="Ana M." last="Bouchet">Ana M. Bouchet</name>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">30023517</idno>
<idno type="pmc">6045331</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045331</idno>
<idno type="RBID">PMC:6045331</idno>
<idno type="doi">10.1021/acsomega.7b00936</idno>
<date when="2017">2017</date>
<idno type="wicri:Area/Pmc/Corpus">000005</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000005</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Singular Interaction between an Antimetastatic Agent and the Lipid Bilayer:
The Ohmline Case</title>
<author><name sortKey="Herrera, Fernando X0a E" sort="Herrera, Fernando X0a E" uniqKey="Herrera F" first="Fernando X0a E." last="Herrera">Fernando X0a E. Herrera</name>
<affiliation><nlm:aff id="aff1">Physics Department,<institution>Universidad Nacional del Litoral, Ciudad Universitaria</institution>
, 3000 Santa Fe,<country>Argentina</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Sevrain, Charlotte M" sort="Sevrain, Charlotte M" uniqKey="Sevrain C" first="Charlotte M." last="Sevrain">Charlotte M. Sevrain</name>
<affiliation><nlm:aff id="aff2"><institution>Université de Brest, CEMCA, UMR CNRS 6521, IBSAM</institution>
, 6, Avenue Victor le Gorgeu, 29238 Brest,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Jaffres, Paul Alain" sort="Jaffres, Paul Alain" uniqKey="Jaffres P" first="Paul-Alain" last="Jaffrès">Paul-Alain Jaffrès</name>
<affiliation><nlm:aff id="aff2"><institution>Université de Brest, CEMCA, UMR CNRS 6521, IBSAM</institution>
, 6, Avenue Victor le Gorgeu, 29238 Brest,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Couthon, Helene" sort="Couthon, Helene" uniqKey="Couthon H" first="Hélène" last="Couthon">Hélène Couthon</name>
<affiliation><nlm:aff id="aff2"><institution>Université de Brest, CEMCA, UMR CNRS 6521, IBSAM</institution>
, 6, Avenue Victor le Gorgeu, 29238 Brest,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Grelard, Axelle" sort="Grelard, Axelle" uniqKey="Grelard A" first="Axelle" last="Grélard">Axelle Grélard</name>
<affiliation><nlm:aff id="aff4"><institution>Université Bordeaux, Institute of Chemistry & Biology of Membranes & Nanoobjects, UMR5248 CNRS</institution>
, Allée de Geoffroy St Hilaire Bât B14 Pessac, 33600 Bordeaux,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Dufourc, Erick J" sort="Dufourc, Erick J" uniqKey="Dufourc E" first="Erick J." last="Dufourc">Erick J. Dufourc</name>
<affiliation><nlm:aff id="aff4"><institution>Université Bordeaux, Institute of Chemistry & Biology of Membranes & Nanoobjects, UMR5248 CNRS</institution>
, Allée de Geoffroy St Hilaire Bât B14 Pessac, 33600 Bordeaux,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chantome, Aurelie" sort="Chantome, Aurelie" uniqKey="Chantome A" first="Aurélie" last="Chantôme">Aurélie Chantôme</name>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Potier Cartereau, Marie" sort="Potier Cartereau, Marie" uniqKey="Potier Cartereau M" first="Marie" last="Potier-Cartereau">Marie Potier-Cartereau</name>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Vandier, Christophe" sort="Vandier, Christophe" uniqKey="Vandier C" first="Christophe" last="Vandier">Christophe Vandier</name>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Bouchet, Ana M" sort="Bouchet, Ana M" uniqKey="Bouchet A" first="Ana M." last="Bouchet">Ana M. Bouchet</name>
<affiliation><nlm:aff id="aff3"><institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">ACS Omega</title>
<idno type="eISSN">2470-1343</idno>
<imprint><date when="2017">2017</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="ao-2017-00936p_0008" id="ab-tgr1"></graphic>
</p>
<p>SK3 channels are
abnormaly expressed in metastatic cells, and Ohmline
(OHM), an ether lipid, has been shown to reduce the activity of SK3
channels and the migration capacity of cancer cells. OHM incorporation
into the plasma membrane is proposed to dissociate the protein complex
formed between SK3 and Orai1, a potassium and a calcium channel, respectively,
and would lead to a modification in the lipid environment of both
the proteins. Here, we report the synthesis of deuterated OHM that
affords the determination, through solid-state NMR, of its entire
partitioning into membranes mimicking the SK3 environment. Use of
deuterated lipids affords the demonstration of an OHM-induced membrane
disordering, which is dose-dependent and increases with increasing
amounts of cholesterol (CHOL). Molecular dynamics simulations comfort
the disordering action and show that OHM interacts with the carbonyl
and phosphate groups of stearoylphosphatidylcholine and sphingomyelin
and to a minor extent with CHOL. OHM is thus proposed to remove the
CHOL OH moieties away from their main binding sites, forcing a new
rearrangement with other lipid groups. Such an interaction takes its
origin at the lipid–water interface, but it propagates toward
the entire lipid molecules and leads to a cooperative destabilization
of the lipid acyl chains, that is, membrane disordering. The consequences
of this reorganization of the lipid phases are discussed in the context
of the OHM-induced inhibition of SK3 channels.</p>
</div>
</front>
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<biblStruct><analytic><author><name sortKey="Nagle, J F" uniqKey="Nagle J">J. F. Nagle</name>
</author>
<author><name sortKey="Tristram Nagle, S" uniqKey="Tristram Nagle S">S. Tristram-Nagle</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article" xml:lang="EN"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">ACS Omega</journal-id>
<journal-id journal-id-type="iso-abbrev">ACS Omega</journal-id>
<journal-id journal-id-type="publisher-id">ao</journal-id>
<journal-id journal-id-type="coden">acsodf</journal-id>
<journal-title-group><journal-title>ACS Omega</journal-title>
</journal-title-group>
<issn pub-type="epub">2470-1343</issn>
<publisher><publisher-name>American Chemical Society</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">30023517</article-id>
<article-id pub-id-type="pmc">6045331</article-id>
<article-id pub-id-type="doi">10.1021/acsomega.7b00936</article-id>
<article-categories><subj-group><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Singular Interaction between an Antimetastatic Agent and the Lipid Bilayer:
The Ohmline Case</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Herrera</surname>
<given-names>Fernando
E.</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
</contrib>
<contrib contrib-type="author" id="ath2"><name><surname>Sevrain</surname>
<given-names>Charlotte M.</given-names>
</name>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="aff3" ref-type="aff">§</xref>
</contrib>
<contrib contrib-type="author" id="ath3"><name><surname>Jaffrès</surname>
<given-names>Paul-Alain</given-names>
</name>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="aff3" ref-type="aff">§</xref>
</contrib>
<contrib contrib-type="author" id="ath4"><name><surname>Couthon</surname>
<given-names>Hélène</given-names>
</name>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="aff3" ref-type="aff">§</xref>
</contrib>
<contrib contrib-type="author" id="ath5"><name><surname>Grélard</surname>
<given-names>Axelle</given-names>
</name>
<xref rid="aff4" ref-type="aff">∥</xref>
</contrib>
<contrib contrib-type="author" id="ath6"><name><surname>Dufourc</surname>
<given-names>Erick J.</given-names>
</name>
<xref rid="aff4" ref-type="aff">∥</xref>
</contrib>
<contrib contrib-type="author" id="ath7"><name><surname>Chantôme</surname>
<given-names>Aurélie</given-names>
</name>
<xref rid="aff3" ref-type="aff">§</xref>
<xref rid="aff5" ref-type="aff">⊥</xref>
</contrib>
<contrib contrib-type="author" id="ath8"><name><surname>Potier-Cartereau</surname>
<given-names>Marie</given-names>
</name>
<xref rid="aff3" ref-type="aff">§</xref>
<xref rid="aff5" ref-type="aff">⊥</xref>
</contrib>
<contrib contrib-type="author" id="ath9"><name><surname>Vandier</surname>
<given-names>Christophe</given-names>
</name>
<xref rid="aff3" ref-type="aff">§</xref>
<xref rid="aff5" ref-type="aff">⊥</xref>
</contrib>
<contrib contrib-type="author" corresp="yes" id="ath10"><name><surname>Bouchet</surname>
<given-names>Ana M.</given-names>
</name>
<xref rid="cor1" ref-type="other">*</xref>
<xref rid="aff3" ref-type="aff">§</xref>
<xref rid="aff5" ref-type="aff">⊥</xref>
</contrib>
<aff id="aff1"><label>†</label>
Physics Department,<institution>Universidad Nacional del Litoral, Ciudad Universitaria</institution>
, 3000 Santa Fe,<country>Argentina</country>
</aff>
<aff id="aff2"><label>‡</label>
<institution>Université de Brest, CEMCA, UMR CNRS 6521, IBSAM</institution>
, 6, Avenue Victor le Gorgeu, 29238 Brest,<country>France</country>
</aff>
<aff id="aff3"><label>§</label>
<institution>Network and Cancer-Canceropole Grand Ouest, (IC-CGO), Maison de la Recherche en Santé</institution>
, 63 Quai Magellan, 44000 Nantes,<country>France</country>
</aff>
<aff id="aff4"><label>∥</label>
<institution>Université Bordeaux, Institute of Chemistry & Biology of Membranes & Nanoobjects, UMR5248 CNRS</institution>
, Allée de Geoffroy St Hilaire Bât B14 Pessac, 33600 Bordeaux,<country>France</country>
</aff>
<aff id="aff5"><label>⊥</label>
Université François Rabelais de Tours, Nutrition, Croissance et Cancer,<institution>Inserm UMR1069</institution>
, 10 Boulevard Tonnellé Bât. Dutrochet, 2ème étage, 37032 Tours,<country>France</country>
</aff>
</contrib-group>
<author-notes><corresp id="cor1"><label>*</label>
E-mail: <email>anamaria.bouchet@univ-tours.fr</email>
(A.M.B.).</corresp>
</author-notes>
<pub-date pub-type="epub"><day>04</day>
<month>10</month>
<year>2017</year>
</pub-date>
<pub-date pub-type="ppub"><day>31</day>
<month>10</month>
<year>2017</year>
</pub-date>
<volume>2</volume>
<issue>10</issue>
<fpage>6361</fpage>
<lpage>6370</lpage>
<history><date date-type="received"><day>06</day>
<month>07</month>
<year>2017</year>
</date>
<date date-type="accepted"><day>08</day>
<month>09</month>
<year>2017</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2017 American Chemical Society</copyright-statement>
<copyright-year>2017</copyright-year>
<copyright-holder>American Chemical Society</copyright-holder>
<license><license-p>This is an open access article published under an ACS AuthorChoice <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/authorchoice_termsofuse.html">License</ext-link>
, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.</license-p>
</license>
</permissions>
<abstract><p content-type="toc-graphic"><graphic xlink:href="ao-2017-00936p_0008" id="ab-tgr1"></graphic>
</p>
<p>SK3 channels are
abnormaly expressed in metastatic cells, and Ohmline
(OHM), an ether lipid, has been shown to reduce the activity of SK3
channels and the migration capacity of cancer cells. OHM incorporation
into the plasma membrane is proposed to dissociate the protein complex
formed between SK3 and Orai1, a potassium and a calcium channel, respectively,
and would lead to a modification in the lipid environment of both
the proteins. Here, we report the synthesis of deuterated OHM that
affords the determination, through solid-state NMR, of its entire
partitioning into membranes mimicking the SK3 environment. Use of
deuterated lipids affords the demonstration of an OHM-induced membrane
disordering, which is dose-dependent and increases with increasing
amounts of cholesterol (CHOL). Molecular dynamics simulations comfort
the disordering action and show that OHM interacts with the carbonyl
and phosphate groups of stearoylphosphatidylcholine and sphingomyelin
and to a minor extent with CHOL. OHM is thus proposed to remove the
CHOL OH moieties away from their main binding sites, forcing a new
rearrangement with other lipid groups. Such an interaction takes its
origin at the lipid–water interface, but it propagates toward
the entire lipid molecules and leads to a cooperative destabilization
of the lipid acyl chains, that is, membrane disordering. The consequences
of this reorganization of the lipid phases are discussed in the context
of the OHM-induced inhibition of SK3 channels.</p>
</abstract>
<custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name>
<meta-value>ao7b00936</meta-value>
</custom-meta>
<custom-meta><meta-name>document-id-new-14</meta-name>
<meta-value>ao-2017-00936p</meta-value>
</custom-meta>
<custom-meta><meta-name>ccc-price</meta-name>
<meta-value></meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body><sec id="sec1"><label>1</label>
<title>Introduction</title>
<p>The
dysregulation of ion channel activity/expression emerged as
a common feature of cancer cells. These dysregulations constitute
new biomarkers<sup><xref ref-type="bibr" rid="ref1">1</xref>
</sup>
and were deeply studied
to better understand the physiology of cancer cells. Interestingly,
the modulation of abnormaly and overexpressed ion channels opens new
perspectives in cancer chemotherapy as illustrated, for instance,
by antiproliferative actions triggering apoptosis<sup><xref ref-type="bibr" rid="ref2">2</xref>
</sup>
or for the reduction of cancer cell spreading.<sup><xref ref-type="bibr" rid="ref3">3</xref>
</sup>
The development of new modulators of ion channels
based on an amphiphilic molecular structure<sup><xref ref-type="bibr" rid="ref4">4</xref>
</sup>
requires knowledge of the mechanisms involved in channel gating
and also their direct interaction with the membrane environment that
is a very complex lipid mixture.<sup><xref ref-type="bibr" rid="ref5">5</xref>
</sup>
</p>
<p>Different mechanisms have been proposed to explain the regulation
of ion channels by lipids. (i) Global change in the physical properties
of the membrane. Analysis of the crystal structure of the KvAP channel
revealed that a lipid bilayer is required to maintain the correct
relative orientations of channel domains<sup><xref ref-type="bibr" rid="ref6">6</xref>
</sup>
and the activity of this voltage-dependent potassium channel also
depends on the negatively charged lipids.<sup><xref ref-type="bibr" rid="ref7">7</xref>
</sup>
TRAAK and TREK1 mechano-gated potassium channels are mechanically
gated by the lipid bilayer in the absence of any other cellular components.<sup><xref ref-type="bibr" rid="ref8">8</xref>
</sup>
Moreover, inverted-conical shape lipids tend
to favor a convex deformation of the plasma membrane which leads to
the opening of the TREK-1/TRAAK channels.<sup><xref ref-type="bibr" rid="ref9">9</xref>
</sup>
It is well-accepted that changes in cholesterol (CHOL) content in
the plasma membrane result in a modification of membrane fluidity
and also bilayer thickness. This bilayer thickness can indeed regulate
the activity of the membrane protein<sup><xref ref-type="bibr" rid="ref10">10</xref>
</sup>
as
exemplified by a reduction of gramicidin channel activity<sup><xref ref-type="bibr" rid="ref11">11</xref>
−<xref ref-type="bibr" rid="ref13">13</xref>
</sup>
induced by an increase of membrane thickness triggered by higher
incorporation of CHOL. (ii) Specific lipid–protein interactions
as exemplified with the interaction between CHOL and the KirBac1.1
channel.<sup><xref ref-type="bibr" rid="ref14">14</xref>
</sup>
(iii) Interactions between channels
and proteins localized in nanodomains of the plasma membrane. Caveolin
which is present in CHOL- and sphingolipid-rich nanodomains was found
to regulate potassium channels.<sup><xref ref-type="bibr" rid="ref15">15</xref>
,<xref ref-type="bibr" rid="ref16">16</xref>
</sup>
This modulation occurred
either through direct protein–lipid interactions<sup><xref ref-type="bibr" rid="ref17">17</xref>
,<xref ref-type="bibr" rid="ref18">18</xref>
</sup>
or by influencing the physical characteristics of the bilayer as
described above.</p>
<p>Ohmline, named hereafter OHM, 1-<italic>O</italic>
-hexadecyl-2-<italic>O</italic>
-methyl-<italic>rac</italic>
-<italic>glycero</italic>
-3-lactose
(<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
), and belonging
to a family of synthetic glyco–glycero ether lipids, has been
demonstrated to specifically reduce the activity of SK3 potassium
channels via an interaction that remains elusive. The SK3 channel
is a potassium channel belonging to a small-conductance calcium-activated
potassium channel family (with SK1 and SK2 channels). When expressed
in a cancer cell, the SK3 channel increases twice the capacity of
cells to migrate and invades a matrix that is similar to the physiological
extracellular matrix.<sup><xref ref-type="bibr" rid="ref19">19</xref>
,<xref ref-type="bibr" rid="ref20">20</xref>
</sup>
Using mouse models of metastatic
breast cancers, the SK3 channel was found to promote the development
of metastases, mainly the development of bone metastases—this
observation has a direct link with the activation of SK3 by calcium
and the high calcium concentrations found in the bone environment.<sup><xref ref-type="bibr" rid="ref21">21</xref>
</sup>
OHM at 10 nM concentration was found to reduce
the SK3 channel activity and the migration of SK3-expressing cancer
cells with 50% efficiency in the bone metastasis development.<sup><xref ref-type="bibr" rid="ref21">21</xref>
</sup>
</p>
<fig id="fig1" position="float"><label>Figure 1</label>
<caption><p>Chemical structure of OHM (1-<italic>O</italic>
-hexadecyl-2-<italic>O</italic>
-methyl-<italic>rac</italic>
-<italic>glycero</italic>
-3-lactose).</p>
</caption>
<graphic xlink:href="ao-2017-00936p_0001" id="gr1" position="float"></graphic>
</fig>
<p>OHM is the first specific inhibitor
of SK3 channels: it inhibits
the SK3 channel and, in a minor extent, the SK1 channel, and it does
not significantly affect the SK2 channel.<sup><xref ref-type="bibr" rid="ref22">22</xref>
</sup>
Altogether, these results provide evidence that amphiphilic compounds
such as OHM can selectively modulate the activity of SK3 channels
likely due to the modification of the physicochemical properties of
the plasma membrane and of the lipid environment even though a direct
interaction of OHM with SK3 channel cannot be excluded.</p>
<p>Despite
the outstanding action of OHM on the SK3 channel activity
and its potent use to prevent the formation of metastases, much less
is known about its interaction with a membrane and even less is known
about the mechanism of action at the molecular level. In this study,
we aimed to assess the behavior of OHM after its incorporation in
a model membrane by answering two basic questions: Does an equilibrium
exist between a location inside and outside the plasma membrane? Are
there any specific interactions with the lipids present in the membrane
that could explain some modification of the membrane biophysical properties?</p>
<p>To answer the first question, we synthesized deuterated OHM at <italic>sn2</italic>
position (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
) and used <sup>2</sup>
H NMR, which allows determining whether
OHM, when placed in contact with a membrane model, features an isotropic
signal (a water environment) or an anisotropic signal (a lipid environment).
To answer the second question, we carried out molecular dynamics (MD)
simulations on a model mimicking the SK3 lipid environment and assessed
membrane dynamic via <sup>2</sup>
H NMR experiments that made use of
deuterated POPC as molecular probe.</p>
<fig id="fig2" position="float"><label>Figure 2</label>
<caption><p>Last two steps for the synthesis of C<sup>2</sup>
H<sub>3</sub>
O-OHM
(<sup>2</sup>
H<sub>3</sub>
-OHM).</p>
</caption>
<graphic xlink:href="ao-2017-00936p_0002" id="gr2" position="float"></graphic>
</fig>
</sec>
<sec id="sec2"><label>2</label>
<title>Results</title>
<sec id="sec2-1"><label>2.1</label>
<title>NMR</title>
<sec id="sec2-1-1"><label>2.1.1</label>
<title>OHM
Partitioning into the Membrane</title>
<p>Wide-line <sup>2</sup>
H NMR
of methyl-deuterated OHM was performed
in water and in POPC/SM/CHOL and POPC membranes, <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
. The spectrum of OHM in solution
(7.6 mM) (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
a,
black trace) is a small so-called “powder” pattern with
two isotropic lines superimposed. One is assigned to residual deuterated
water (centered at 4.7 ppm), and the other one is assigned to OHM
in solution (at 2.3 ppm). Solid-state <sup>2</sup>
H NMR is indeed
capable of distinguishing molecules in solution, in small isotropic
micelles, or in larger entities. Fast molecular tumbling as in solution
or for nanometric micelles leads to the so-called isotropic lines;
the solid-state quadrupolar interaction is averaged to zero by fast
(picosecond) Brownian motions.<sup><xref ref-type="bibr" rid="ref23">23</xref>
</sup>
For larger
entities, micrometric liposomes or large vesicles, the slow motions
(microseconds to nanoseconds), mainly anisotropic, do not average
the interaction to zero. One obtains residual “powder patterns”
reflecting the onset of such slow motions. An intermediate situation
may be obtained for edifices of a 50–200 nm hydrodynamic radius,
such as wormlike micelles, which present “exchange”
line shapes<sup><xref ref-type="bibr" rid="ref23">23</xref>
</sup>
as observed for the very
small residual powder pattern in <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
a. The experimental spectrum could be simulated
(red traces and inset) using a quadrupolar splitting of 2.6 kHz and
two isotropic lines of 29 and 13% contributing to water and OHM, respectively,
in solution. The presence of such a powder pattern (58%) indicated
that deuterated OHM is under the form of large micelles (possibly
wormlike). When applied to liposomes, the spectrum of <sup>2</sup>
H OHM is composed of a central isotropic line at 4.7 ppm (deuterated
water) and a well-defined axially symmetric powder pattern representative
of entire partitioning of OHM into the membrane (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
b); signals of OHM in micelles
or in solution are no longer detected (less than 1% experimental error).
Such patterns are characteristic of molecules embedded in liquid-disordered
or liquid-ordered (lo) environments that maintain the axial symmetry
of the motional processes occurring under such conditions. Simulations
(red traces) report quadrupolar splittings of 9.2 and 7.4 kHz for
OHM in POPC/SM/CHOL and POPC liposomes, respectively. This indicates
that OHM sits in a more ordered environment when embedded in a CHOL-containing
membrane (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
b).</p>
<fig id="fig3" position="float"><label>Figure 3</label>
<caption><p><sup>2</sup>
H NMR spectra of deuterated OHM (a) in water (7.6 mM),
(b) in POPC/SM/CHOL (10/60/30) at Ri = 15, and (c) in POPC at Ri =
15. Black traces stand for the experimental spectra, and red traces
stand for the simulated ones. The top inset is the expansion of the
simulation of (a), with W, i, and m representing water, isotropic,
and micelle traces of OHM.</p>
</caption>
<graphic xlink:href="ao-2017-00936p_0003" id="gr3" position="float"></graphic>
</fig>
</sec>
<sec id="sec2-1-2"><label>2.1.2</label>
<title>OHM Disorders Membranes</title>
<p>The effect
of OHM on the dynamics of several membrane systems was monitored using
POPC perdeuterated on the palmitic <italic>sn</italic>
-1 chain. POPC
was used either as a pure system or in the presence of major amounts
of sphingomyelin (SGML), that is, POPC/SM/CHOL (10/85/5, mol %) or
with a high concentration of CHOL, that is, POPC/SM/CHOL (10/60/30,
mol %). The temperature was varied from 25 to 45 °C on all systems.
Spectra are recorded at 45 °C in <xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
(left) and in the presence of OHM at lipid-to-OHM
ratio, Ri, of 30 (right). All spectra display the axially symmetric
shape, characteristic of the manifestation of several axially symmetric
motional processes (bond or molecule rotation, anisotropic reorientations,
etc.) known to be present in rather dynamic membranes.<sup><xref ref-type="bibr" rid="ref24">24</xref>
,<xref ref-type="bibr" rid="ref25">25</xref>
</sup>
The pure POPC system is the most dynamic membrane (narrow spectrum).
The presence of high amounts of SGML and 5 mol % CHOL widens the spectra
(middle traces), which indicates a reduction in membrane dynamics.
With higher amounts of CHOL (POPC/SM/CHOL, 10/60/30, mol %), the spectra
become very wide, indicating further reduction of dynamics. In the
presence of OHM, the spectral shapes are unchanged but become a little
narrower. This is better perceived when performing spectral simulations
(red traces below the experimental spectra) and reporting the <italic>S</italic>
<sup>CD</sup>
order parameter measured at each labeled
carbon position, <italic>k</italic>
, on the palmitic chain of POPC
(<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
, right).
All profiles display the well-known behavior: “plateau”
of elevated order parameters near the very rigid glycerol backbone
(positions 2–8) and monotonic decrease on moving toward the
chain end at position 16, that is, at the bilayer center.<sup><xref ref-type="bibr" rid="ref24">24</xref>
</sup>
The presence of OHM is almost undetectable on
POPC, slightly decreases the whole profile of order parameters for
POPC/SM/CHOL (10/85/5), and has more disordering action on that for
POPC/SM/CHOL (10/60/30). By summing up all order parameters along
the chain, the effect is of 1 ± 1% for POPC, 3 ± 1% for
POPC/SM/CHOL (10/85/5), and 5 ± 1% for POPC/SM/CHOL (10/60/30).
The disordering action is concentration-dependent: upon increasing
the amount of OHM (Ri = 15, not shown) on the POPC/SM/CHOL (10/60/30)
membrane, the whole disordering becomes 8%. Of interest is the observation
that disordering is observed for temperatures in the range 35–45
°C; no effect on membrane dynamics is detected at ambient temperature.
It is worth mentioning that membrane disordering is synonymous of
membrane thinning. OHM thus leads to a reduction of membrane thickness;
the more OHM, the greater the effect and the more CHOL in the membrane,
the greater the effect.</p>
<fig id="fig4" position="float"><label>Figure 4</label>
<caption><p>Left panel: solid-state <sup>2</sup>
H NMR spectra
in the absence
(a–c) and the presence (d–f) of OHM (Ri = 30), at 45
°C: (a) <sup>2</sup>
H<sub>31</sub>
-POPC, (b) <sup>2</sup>
H<sub>31</sub>
-POPC/SM/CHOL (10/85/5, mol %), and (c) <sup>2</sup>
H<sub>31</sub>
-POPC/SM/CHOL (10/60/30, mol %). Black upper traces represent
the experimental spectra, and red lower traces represent the simulated
spectra. Right panel: order parameter profile |<italic>S</italic>
<sub><italic>k</italic>
</sub>
<sup arrange="stack">CD</sup>
| as a function of <italic>k</italic>
th labeled carbon position,
of the palmitic chain of POPC in the absence (black symbols, a–c)
and the presence (purple symbols, d–f) of OHM at Ri = 30. |<italic>S</italic>
<sub><italic>k</italic>
</sub>
<sup arrange="stack">CD</sup>
| is obtained from spectral simulations, with
arbitrary decremental assignment for positions 2–10. The error
bars represent the accuracy.</p>
</caption>
<graphic xlink:href="ao-2017-00936p_0004" id="gr4" position="float"></graphic>
</fig>
</sec>
</sec>
<sec id="sec2-2"><label>2.2</label>
<title>MD Simulation</title>
<sec id="sec2-2-1"><label>2.2.1</label>
<title>OHM
Position in the Bilayer and the 2D Order
Parameter</title>
<p>From MD simulations, information on membrane dynamics
and the location of OHM in the bilayer can be obtained. OHM is located
inside the bilayer, with its sugar groups pointing at the water interface
and the hydrocarbon chain well-inserted in the bilayer aliphatic core.
This is well-shown in the density profile for the final arrangement
of the OHM molecules along the <italic>z</italic>
axis of the bilayer
(<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">Figure S12</ext-link>
).</p>
<p><xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref>
</xref>
shows the 2D lipid tail order parameters
calculated at different distances from the center of the membrane
(slices at different depths) and their correlation with the OHM molecule
position. In the control bilayer (the first column), there is a clear
difference in the surface order parameter between the interface at
14–16 Å and the hydrocarbon core region at 8–10
Å and the bilayer center at 0–2 Å. The center is
clearly more disordered than the two other bilayer regions, in complete
accordance with the above-mentioned NMR data. The bilayer with OHM
(the second column) shows, for the regions at 8–10 and 14–16
Å, a decrease in the surface order parameter for regions that
match with the OHM positions that are shown in the third column. Of
interest is the fact that the region around 8–10 Å appears
to be the most ordered, as was also noted in the NMR experimental
order parameters (positions 2–10 plateau along the fatty acyl
chains). <italic>S</italic>
<sup>CD</sup>
acyl chain order parameters
can also be obtained from MD calculations: we have obtained results
in the same line of the NMR experiments, and the presence of ≈10%
of OHM also induces a decrease in order parameters of about 10% (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">Figure S13</ext-link>
).</p>
<fig id="fig5" position="float"><label>Figure 5</label>
<caption><p>Two-dimensional order parameter in the
lipid model system. Total
order parameter of the upper leaflet of the bilayer is represented
without and with OHM. The position of the OHM molecules for the same
leaflet is indicated in 2D-OHM position. The numbers in angstrom on
the left indicate the distances from the center of the membrane at
which the 2D order parameter was evaluated. The bilayer center is
set at 0 Å.</p>
</caption>
<graphic xlink:href="ao-2017-00936p_0005" id="gr5" position="float"></graphic>
</fig>
</sec>
<sec id="sec2-2-2"><label>2.2.2</label>
<title>Consequences
of the OHM Partitioning: Interleaflet
Mixing</title>
<p>The order parameter decrease is usually associated
with an increase in the bilayer interdigitation and its related diminution
in the thickness. The interleaflet distributions of the methyl group
ends of the acyl chains are measures of the mixing between the opposing
monolayers.<sup><xref ref-type="bibr" rid="ref26">26</xref>
</sup>
The density profile of OHM
(<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">Figure S12</ext-link>
, bottom row) shows the interdigitation
of the acyl chains.</p>
<p>Distances between the peaks, which characterize
the distributions of the terminal methyl groups in both leaflets,
and the percentage of the terminal methyl groups from each monolayer
shared by the opposite (overlap) are shown in <xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref>
</xref>
.</p>
<table-wrap id="tbl1" position="float"><label>Table 1</label>
<caption><title>Interleaflet Mixing<xref rid="t1fn1" ref-type="table-fn">a</xref>
</title>
</caption>
<table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col>
<col align="left"></col>
<col align="char" char="±"></col>
<col align="left"></col>
</colgroup>
<thead><tr><th style="border:none;" align="center"> </th>
<th style="border:none;" align="center">CH<sub>3</sub>
source</th>
<th style="border:none;" align="center" char="±">distance between peaks (Å)</th>
<th style="border:none;" align="center">overlap
(%)</th>
</tr>
</thead>
<tbody><tr><td style="border:none;" align="left">RAFT</td>
<td style="border:none;" align="left">PC</td>
<td style="border:none;" align="char" char="±">2.3 ± 0.4</td>
<td style="border:none;" align="left">∼59.6</td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">SGML</td>
<td style="border:none;" align="char" char="±">5.3 ± 0.5</td>
<td style="border:none;" align="left">∼25.4</td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">CHOL</td>
<td style="border:none;" align="char" char="±">4.1 ± 0.4</td>
<td style="border:none;" align="left">∼30.4</td>
</tr>
<tr><td style="border:none;" align="left">RAFT + OHM</td>
<td style="border:none;" align="left">PC</td>
<td style="border:none;" align="char" char="±">3.2 ± 0.7</td>
<td style="border:none;" align="left">∼60.7</td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">SGML</td>
<td style="border:none;" align="char" char="±">5.2 ± 0.7</td>
<td style="border:none;" align="left">∼41.6</td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">CHOL</td>
<td style="border:none;" align="char" char="±">3.2 ± 0.6</td>
<td style="border:none;" align="left">∼55.8</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t1fn1"><label>a</label>
<p>The interleaflet mixing was calculated
as the overlap area between the atomic density profiles of the terminal
CH<sub>3</sub>
groups coming from both monolayers and by the distance
between the peaks of such densities.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>The overlap in the distributions of the CH<sub>3</sub>
terminal
groups increases after OHM addition for all three lipids [1,2-distearoyl-<italic>sn</italic>
-glycero-3-phosphocholine (DSPC), SGML, and CHOL]. For
DSPC, a slight increase in the overlap is observed (1.1%); for SGML
and CHOL molecules, instead the increase in the overlap is more significant
(16.2 and 25.4%, respectively). The analysis confirms that the OHM
molecules induce a change at the level of the interlayer space with
small consequences in terms of average area per lipid and thickness,
+0.2 Å<sup>2</sup>
and −1.6 Å, respectively.</p>
</sec>
<sec id="sec2-2-3"><label>2.2.3</label>
<title>Molecular Interactions at the Interface
That Stabilize OHM</title>
<p>To characterize the intermolecular interactions
that stabilize OHM at the interface level, the cumulative number of
molecules located around the different atomic moieties of the lipids
is plotted for each lipid group of the bilayer as well as the water
molecules (<xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref>
</xref>
). The <italic>sn</italic>
3 oxygen atom of the OHM glycerol group
that links the lipid chain (noted O1, <xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
) is located at the water–lipid interface,
and we have chosen it as a reference (0 Å). In each inset, the
radial distribution functions (RDFs) of the lipid groups considered
are drawn.</p>
<fig id="fig6" position="float"><label>Figure 6</label>
<caption><p>Cumulative number of molecules and RDFs of OHM oxygen glycerol
associated with respect to the main lipid groups located at the interface.
The RDFs for the different membrane groups around one OHM oxygen atom,
between the tail and the sugar head [oxygen O1 of the glycerol moiety
(<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
)], are shown
in the inset. The colors indicate the lipid considered, and in each
graph, the moiety involved is indicated. The origin of the graphs
at 0 Å is not shown for clarity.</p>
</caption>
<graphic xlink:href="ao-2017-00936p_0006" id="gr6" position="float"></graphic>
</fig>
<p>From the first rising region of the cumulative number and
RDF curves,
the first groups that appear nearest to the OHM oxygen atom O1 (at
around 0.3 nm) are the carbonyl groups of DSPC and SGML; the CHOL
hydroxyl and water molecules; and then phosphates, amides, and cholines.
Nevertheless, only the carbonyl and phosphorus atoms from DSPC seem
to be ordered at around 0.5 nm from the location of the OHM oxygen
O1 because only both of these atoms show a well-defined peak in the
RDF curve. <xref rid="tbl2" ref-type="other">Table <xref rid="tbl2" ref-type="other">2</xref>
</xref>
allows
the comparison of the number of atoms that are located, on average,
at a specific distance of 0.8 nm from the OHM oxygen O1.</p>
<table-wrap id="tbl2" position="float"><label>Table 2</label>
<caption><title>Summary of the Cumulative Number of
Different Lipid Species and Their Corresponding Atomic Groups at 0.8
nm Distance for OHM Oxygen O1</title>
</caption>
<table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col>
<col align="left"></col>
<col align="left"></col>
<col align="left"></col>
</colgroup>
<thead><tr><th style="border:none;" align="center"> </th>
<th style="border:none;" align="center">DSPC</th>
<th style="border:none;" align="center">SGML</th>
<th style="border:none;" align="center">CHOL</th>
</tr>
</thead>
<tbody><tr><td style="border:none;" align="left">O carbonyl</td>
<td style="border:none;" align="left">1.63</td>
<td style="border:none;" align="left">0.63</td>
<td style="border:none;" align="left"> </td>
</tr>
<tr><td style="border:none;" align="left">P atoms</td>
<td style="border:none;" align="left">0.3</td>
<td style="border:none;" align="left">0.04</td>
<td style="border:none;" align="left"> </td>
</tr>
<tr><td style="border:none;" align="left">N choline</td>
<td style="border:none;" align="left">0.09</td>
<td style="border:none;" align="left">0.05</td>
<td style="border:none;" align="left"> </td>
</tr>
<tr><td style="border:none;" align="left">N amide</td>
<td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">0.2</td>
<td style="border:none;" align="left"> </td>
</tr>
<tr><td style="border:none;" align="left">O chol</td>
<td style="border:none;" align="left"> </td>
<td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">0.4</td>
</tr>
<tr><td style="border:none;" align="left">waters
around oxygen OHM</td>
<td colspan="3" align="left">18</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>The OHM molecules interact
mostly with the carbonyl oxygen of DSPC
molecules, followed by the carbonyl oxygen of SGML and finally the
CHOL oxygen. The strong solvation of the OHM molecules, principally
at the level of the sugar head, suggests that the water molecules
could be playing a role in the stabilization of the head group through
hydrogen interactions with other lipid head groups.</p>
<p>We also
calculated the RDFs of the different lipid moieties around
the CHOL oxygen (<xref rid="fig7" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref>
</xref>
). The RDFs show that CHOL is stabilized by the DSPC as well as SGML
carbonyls (upper row). The presence of a sharp peak centered at 0.27
nm indicates that a strong H bond is formed between the hydroxyl and
the carbonyl groups.</p>
<fig id="fig7" position="float"><label>Figure 7</label>
<caption><p>RDFs of CHOL hydroxyl. The RDFs for the different membrane
groups
(black for DSPC and red for SGML) around the CHOL oxygen are shown.</p>
</caption>
<graphic xlink:href="ao-2017-00936p_0007" id="gr7" position="float"></graphic>
</fig>
<p>On the basis of the peak heights,
in the control bilayer, there
are equal possibilities to find a carbonyl oxygen atom from DSPC or
SGML at around 0.27 nm. However, in the presence of OHM, the possibility
increases (around 50%) for the SGML carbonyl oxygen. For the interactions
of the CHOL oxygen with the choline nitrogen (middle row), the OHM
molecule has little effect and the possibilities to find it at around
0.4 nm are maintained after the addition of OHM. Finally, for the
phosphorus atom, the possibility to find it at 0.4 nm from the CHOL
oxygen is higher for the DSPC molecule than that for SGML (lower left
row). Because of the addition of OHM, the CHOL hydroxyl again misses
some of these interactions.</p>
</sec>
</sec>
</sec>
<sec id="sec3"><label>3</label>
<title>Discussion</title>
<p>Over the last 10 years, the role of ion channels in cancer development
and cancer spreading was deeply assessed,<sup><xref ref-type="bibr" rid="ref4">4</xref>
,<xref ref-type="bibr" rid="ref27">27</xref>
</sup>
which
paved the way for the design of efficient inhibitors. In this direction,
the implication of SK3 channels in cancer cell migration and metastasis
development was already established.<sup><xref ref-type="bibr" rid="ref19">19</xref>
</sup>
Edelfosine<sup><xref ref-type="bibr" rid="ref28">28</xref>
</sup>
and OHM<sup><xref ref-type="bibr" rid="ref21">21</xref>
</sup>
were the
first amphiphilic compounds that exhibited strong SK3 inhibition.
Although more works were reported to conceive heterocyclic compounds
as inhibitors of SK3 channels,<sup><xref ref-type="bibr" rid="ref29">29</xref>
</sup>
the fact
that amphiphilic compounds can also act as efficient modulators offers
new possibilities of assessment. These original results invited to
study the mechanism of action of OHM that was first addressed by designing
new analogues (structure/activity relationship). Accordingly, analogues
of OHM featuring the incorporation of a phosphate group or other disaccharide
unit<sup><xref ref-type="bibr" rid="ref30">30</xref>
</sup>
gave new efficient inhibitors, but
OHM remains the most promising compound. Accordingly, further insights
into the mechanism of action of amphiphilic compounds on ion channels
require the assessment of their interactions with model membranes,
which is the aim of our study.</p>
<p>From the NMR experiments, two
main pieces of information can be
derived: OHM is partitioned entirely into the membrane and disorders
the lipid bilayer. The first information is of interest because it
implies that there is a greater affinity for the lipid bilayer compared
to the self-association of OHM as a micelle in water. The OHM critical
micelle concentration value of 12 μM<sup><xref ref-type="bibr" rid="ref31">31</xref>
</sup>
is hundreds of times lower than the values used in the experiments.
The composition of the membrane appears not to be of importance; pure
phospholipids or mixtures with sphingolipids and CHOL still lead to
100% of OHM entering into liposomes both at Ri = 15 and 30. We have,
however, to be careful because no accurate determination of binding
constants has been performed. The second important observation is
the disordering effect observed on membranes containing large amounts
of CHOL. The system POPC/SM/CHOL (10/60/30) is indeed in a lo phase
where molecular motions are clearly reduced by the condensing effect
of CHOL. The action of OHM appears to limit the action of CHOL by
reducing the condensing effect.</p>
<p>From MD (<xref rid="fig5" ref-type="fig">Figures <xref rid="fig5" ref-type="fig">5</xref>
</xref>
and <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">S13</ext-link>
), we
apprehend that the disordering
effect can be attributed to distinct mechanisms, one involving a direct
interaction of the OHM molecules with both species (DSPC and SGML)
or another involving a CHOL-mediated interaction, affecting the binding
of those lipids with the CHOL molecules because it was already mentioned
that CHOL molecules induce an ordering effect on the lipid tails.
A combination of both mechanisms cannot be excluded.</p>
<p>For edelfosine
and miltefosine, a tendency was proposed to be associated
with CHOL in certain lipid domains.<sup><xref ref-type="bibr" rid="ref4">4</xref>
,<xref ref-type="bibr" rid="ref32">32</xref>
</sup>
Other ether
lipids have been demonstrated to force the CHOL OH to interact with
the phosphate lipid group due to the absence of carbonyl, to be stabilized.<sup><xref ref-type="bibr" rid="ref33">33</xref>
</sup>
The consequence of this new stabilization promoted
a tilt in the CHOL molecule that the same author has mentioned as
a modulator of membrane-active proteins. In our model, the latter
would seem to be the case (<xref rid="fig6" ref-type="fig">Figures <xref rid="fig6" ref-type="fig">6</xref>
</xref>
and <xref rid="fig7" ref-type="fig">7</xref>
).</p>
<p>CHOL controls
the activity of a wide range of membrane proteins
through specific interactions.<sup><xref ref-type="bibr" rid="ref34">34</xref>
</sup>
Indeed,
protein sequence analysis revealed that several sites of SK3 are CHOL-binding
candidates (not shown). The OHM-induced modifications of CHOL availability
in the bilayer could modify its interaction with regions that recognize
CHOL, leading to an incorrect or inclusive misfolding of the SK3 structure
with loss of activity. This mechanism of decrease/loss of activity
(inactivation is a particular state for a channel that occurs after
activation) does, however, not exclude the fact that OHM provokes
a general destabilization of the acyl chains that would also affect
the activity of the channel.</p>
<p>So far, it was not demonstrated
that SK3 is a lipid-dependent channel,
but the channel is activated by cell swelling and inhibited by shrinkage,<sup><xref ref-type="bibr" rid="ref35">35</xref>
</sup>
which suggests that the channel activity is
sensitive to the biophysical characteristics of the plasma membrane.
A change in the global organization of the lipid environment could
explain the modulation of the SK3 channel activity, but a very specific
lipid interaction should not be excluded.</p>
<p>The results in <xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref>
</xref>
show that the presence
of OHM induces an increase in the interleaflet
mixing. It should be noticed that the length of the hydrophobic alkyl
chain was described as a key characteristic for the activity of alkylphospholipid
derivatives that have been used as precursors to design the OHM molecules.
As shown previously for glycerol-derived phospholipid ether analogues,
the decrease in the length from 12 carbons to 7 resulted in little
or no anticancer activity, whereas increasing the length had the opposite
effect.<sup><xref ref-type="bibr" rid="ref36">36</xref>
</sup>
For the OHM case, this behavior
is in complete agreement with our preliminary experimental data (unpublished
results), meaning that as a result of the OHM internalization or as
a driving force to pull the hydrophilic moiety at the interface, the
interleaflet mixing (the mixing between methyl group ends of the acyl
chains from opposite monolayers) should be considered in the mechanism
of action of this kind of compound.</p>
</sec>
<sec id="sec4"><label>4</label>
<title>Conclusions</title>
<p>This work brings new insights into the mechanism of action of OHM
that, as previously shown, can inhibit (in vitro and in vivo) the
activity of SK3 ion channels. We demonstrate, for the first time,
that deuterated OHM (<sup>2</sup>
H<sub>3</sub>
-OHM) is fully incorporated
in a model of membrane as revealed by <sup>2</sup>
H NMR studies. Additional <sup>2</sup>
H NMR studies that make use of model membranes that incorporate
perdeuterated POPC indicate that the addition of OHM has a weak effect
on the ordering parameters within the bilayer. However, model membranes
with a high concentration of CHOL (30%) or the recording around the
physiological temperature (35–45 °C instead of 20 °C)
shows a more pronounced effect of OHM on the behavior of the bilayer
that becomes less ordered. These results indicate that OHM can slightly
modify the physicochemical properties of the bilayer, especially the
moieties rich in CHOL. The question of the location of OHM in this
lipid bilayer is then addressed by MD simulations. From this study,
it is concluded that OHM mainly interacts with the oxygen atoms of
the carbonyl groups of DSPC and SGML and, in a less extent, with the
oxygen atom of CHOL. The consequences of these interactions imply
a reduction of the stabilization of CHOL within the membrane that,
in the absence of OHM, interacts also with the oxygen atoms of the
carbonyl groups of DSPC and SGML. These results show, for the first
time, the molecular interactions between OHM and model membranes.
The presence of direct interaction between OHM embedded in the lipid
bilayer and ion channels cannot be so far excluded. This point still
needs to be studied and is the focus of our current efforts.</p>
</sec>
<sec id="sec5"><label>5</label>
<title>Materials and Methods</title>
<sec id="sec5-1"><label>5.1</label>
<title>Synthesis of Deuterated
OHM at the <italic>sn</italic>
2 Position</title>
<p>The full synthesis
is reported in
the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">Supporting Information</ext-link>
. The last two
steps (glycosylation and deprotection of the lactose unit) are reported
below.</p>
<sec id="sec5-1-1"><label>5.1.1</label>
<title>Glycosylation Reaction</title>
<p>A solution
of <bold>1</bold>
(459 mg, 0.59 mmol, 1.0 equiv) and <bold>2</bold>
(200 mg, 0.60 mmol, 1.02 equiv) in dry CH<sub>2</sub>
Cl<sub>2</sub>
(10 mL) was stirred with molecular sieves (4 Å) for 1 hour
under a N<sub>2</sub>
atmosphere. At 0 °C, BF<sub>3</sub>
·Et<sub>2</sub>
O (29 μL, 0.235 mmol, 0.4 equiv) was added dropwise,
and the mixture was stirred for 24 h at room temperature under inert
atmosphere. The mixture was quenched by the addition of water (3 mL).
The organic layer was washed twice with an aqueous saturated NaHCO<sub>3</sub>
solution (2 × 3 mL) and an aqueous saturated NaCl solution
(3 mL). The organic layer was dried upon MgSO<sub>4</sub>
, filtered,
and concentrated to give the crude compound <bold>3</bold>
. The product
was purified by chromatography on silica gel [eluent: petroleum spirit/ethyl
acetate (6:4)] to give the pure compound <bold>3</bold>
(42% yield).
Rf [petroleum spirit/ethyl acetate (6:4)]: 0.12; <sup>1</sup>
H NMR
(C<sup>2</sup>
HCl<sub>3</sub>
, 399.972): 5.32 (d, 1H, <sup>3</sup>
<italic>J</italic>
<sub>HH</sub>
= 3.2 Hz, <italic>H</italic>
<sub><italic>4</italic>
′</sub>
); 5.16 (t, 1H, <sup>3</sup>
<italic>J</italic>
<sub>HH</sub>
= 8.8 Hz, H<sub>3</sub>
); 5.08 (dd, 1H, <sup>3</sup>
<italic>J</italic>
<sub>HH</sub>
= 10.0, 7.6 Hz, H<sub>2′</sub>
); 4.94–4.86 (m, 2H, H<sub>2</sub>
+ H<sub>3′</sub>
); 4.52–4.44 (m, 3H, H<sub>1</sub>
+ H<sub>1′</sub>
+ H<sub>6a</sub>
); 4.10–4.05 (m, 3H, H<sub>6′a</sub>
+ H<sub>6′b</sub>
+ H<sub>6b</sub>
); 3.86–3.79 (m,
2H, H<sub>5′</sub>
+ <italic>Ha</italic>
C<italic>H</italic>
<sub><italic>2</italic>
</sub>
<italic>sn-3</italic>
); 3.79 (t, 1H, <sup>3</sup>
<italic>J</italic>
<sub>HH</sub>
= 9.4 Hz, H<sub>4</sub>
);
3.58–3.56 (m, 2H, <italic>Ha</italic>
C<italic>H</italic>
<sub><italic>2</italic>
</sub>
<italic>sn-1</italic>
+ <italic>Hb</italic>
C<italic>H</italic>
<sub><italic>2</italic>
</sub>
<italic>sn-3</italic>
); 3.43–3.35 (m, 5H, H<sub>5</sub>
+ C<italic>H sn-2</italic>
+ C<italic>H</italic>
<sub><italic>2</italic>
</sub>
α fatty
chain + <italic>Hb</italic>
C<italic>H</italic>
<sub><italic>2</italic>
</sub>
<italic>sn-1</italic>
); 2.12 (s, 3H, OC<italic>H</italic>
<sub><italic>3</italic>
</sub>
); 2.09 (s, 3H, OC<italic>H</italic>
<sub><italic>3</italic>
</sub>
); 2.03 (s, 3H, OC<italic>H</italic>
<sub><italic>3</italic>
</sub>
); 2.01 (s, 3H, OC<italic>H</italic>
<sub><italic>3</italic>
</sub>
); 2.01 (s, 3H, OC<italic>H</italic>
<sub><italic>3</italic>
</sub>
); 2.00 (s, 3H, OC<italic>H</italic>
<sub><italic>3</italic>
</sub>
); 1.93 (s, 3H, OC<italic>H</italic>
<sub><italic>3</italic>
</sub>
); 1.52 (m, 2H, β C<italic>H</italic>
<sub><italic>2</italic>
</sub>
fatty chain); 1.22 (br s, 26H, C<italic>H</italic>
<sub><italic>2</italic>
</sub>
fatty chain); 0.86 (t, 3H, <sup>3</sup>
<italic>J</italic>
<sub>HH</sub>
= 6.6 Hz, C<italic>H</italic>
<sub><italic>3</italic>
</sub>
fatty chain); <sup>13</sup>
C NMR (CDCl<sub>3</sub>
, 74.475): 170.3 (s, <italic>C</italic>
=O); 170.1 (s, <italic>C</italic>
=O); 170.0 (s, <italic>C</italic>
=O); 169.7
(s, <italic>C</italic>
=O); 169.5 (s, <italic>C</italic>
=O);
169.0 (s, <italic>C</italic>
=O); 101.0 (s, C<sub>1′</sub>
); 100.9–100.8 (C<sub>1</sub>
two diastereoisomers); 79.2–78.8
(<italic>C</italic>
H <italic>sn-2</italic>
, two diastereoisomers);
76.2 (s, C<sub>4</sub>
); 72.8 (s, <italic>C</italic>
<sub>3</sub>
);
72.6 (s, <italic>C</italic>
<sub>5</sub>
); 71.8 (<italic>C</italic>
H<sub>2</sub>
α fatty chain); 71.7 (s, <italic>C</italic>
<sub>2</sub>
); 71.0 (<italic>C</italic>
<sub>3′</sub>
); 70.6 (<italic>C</italic>
<sub>5′</sub>
); 70.3–70.0 (<italic>C</italic>
H<sub>2</sub>
<italic>sn-3</italic>
, two diastereoisomers); 69.8–68.8
(<italic>C</italic>
H<sub>2</sub>
<italic>sn-1</italic>
, two diastereoisomers);
69.1 (s, <italic>C</italic>
<sub>2′</sub>
); 66.6 (s, <italic>C</italic>
<sub>4′</sub>
); 62.0 (s, <italic>C</italic>
<sub>6</sub>
); 60.8 (s, <italic>C</italic>
<sub>6′</sub>
); 31.9
(s, <italic>C</italic>
H<sub>2</sub>
fatty chain); 29.4 (s, <italic>C</italic>
H<sub>2</sub>
fatty chain); 29.3 (s, <italic>C</italic>
H<sub>2</sub>
fatty chain); 26.0 (s, <italic>C</italic>
H<sub>2</sub>
fatty chain); 22.6 (s, <italic>C</italic>
H<sub>2</sub>
fatty chain);
20.8 (s, O<italic>C</italic>
H<sub>3</sub>
); 20.6 (s, O<italic>C</italic>
H<sub>3</sub>
); 20.5 (s, O<italic>C</italic>
H<sub>3</sub>
); 14.1
(s, <italic>C</italic>
H<sub>3</sub>
fatty chain).</p>
</sec>
<sec id="sec5-1-2"><label>5.1.2</label>
<title>Deprotection of <bold>3</bold>
to Produce <sup>2</sup>
H<sub>3</sub>
-OHM</title>
<p>K<sub>2</sub>
CO<sub>3</sub>
(2.8
mL, 0.02 mmol, 0.5 equiv) was added to a solution of <bold>3</bold>
(40 mg, 0.10 mmol, 1.0 equiv) in MeOH (5 mL). The mixture was stirred
at room temperature for 15 h. Then, Amberlyst IR-120 (H<sup>+</sup>
) was added, and the mixture was stirred for 30 min at room temperature.
The reaction mixture was warmed (reflux), quickly filtered, and concentrated
to give the crude compound <sup>2</sup>
H<sub>3</sub>
-OHM in a quantitative
yield. <sup>2</sup>
H NMR (deuterium NMR probehead): 3.78 (s, C<sup><italic>2</italic>
</sup>
<italic>H</italic>
<sub><italic>3</italic>
</sub>
); <sup>1</sup>
H NMR (DMSO-<sup><italic>2</italic>
</sup>
<italic>H</italic>
<sub><italic>6</italic>
</sub>
, 500.133): 5.11–5.07
(m, 2H, 2O<italic>H</italic>
); 4.77 (br s, 1H, O<italic>H</italic>
); 4.66 (s, 1H, O<italic>H</italic>
); 4.63 (br s, 1H, O<italic>H</italic>
); 4.53–4.50 (m, 2H, H<sub>1′</sub>
+ O<italic>H</italic>
); 4.20–4.17 (m, 2H, H<sub>4</sub>
+ H<sub>1′</sub>
); 3.77–3.26 (m, 17H, H<sub>3</sub>
+ H<sub>5</sub>
+ H<sub>6</sub>
+ H<sub>2′</sub>
+ H<sub>3′</sub>
+ H<sub>4′</sub>
+ H<sub>5′</sub>
+ H<sub>6′</sub>
+ C<italic>H</italic>
<sub><italic>2</italic>
</sub>
α fatty alkyl chain + C<italic>H</italic>
<sub><italic>2</italic>
</sub>
<italic>sn-1</italic>
+ C<italic>H</italic>
<sub><italic>2</italic>
</sub>
<italic>sn-3</italic>
+ C<italic>H sn-2</italic>
); 3.00–2.99 (m, 1H, H<sub>2</sub>
); 1.47 (m,
2H, β C<italic>H</italic>
<sub><italic>2</italic>
</sub>
fatty
alkyl chain); 1.22 (br s, 26H, C<italic>H</italic>
<sub><italic>2</italic>
</sub>
fatty alkyl chain); 0.85 (t, 3H, <sup>3</sup>
<italic>J</italic>
<sub>HH</sub>
= 6.8 Hz, C<italic>H</italic>
<sub><italic>3</italic>
</sub>
fatty alkyl chain); <sup>13</sup>
C NMR (C<sup>2</sup>
HCl<sub>3</sub>
, 74.475): 103.8 (s, C<sub>1′</sub>
); 102.9–102.8
(C<sub>1</sub>
two diastereoisomers); 80.7 (C<sub>4</sub>
); 78.6–78.5
(<italic>C</italic>
H <italic>sn-2</italic>
, two diastereoisomers);
75.5; 75.0; 74.8; 73.2; 73.1; 70.5; 68.1 (C<sub>2</sub>
+ C<sub>3</sub>
+ C<sub>5</sub>
+ C<sub>2′</sub>
+ C<sub>3′</sub>
+
C<sub>4′</sub>
+ C<sub>5′</sub>
); 70.6 (<italic>C</italic>
H<sub>2</sub>
α fatty alkyl chain); 69.9 (<italic>C</italic>
H<sub>2</sub>
<italic>sn-3</italic>
); 68.7–68.5 (<italic>C</italic>
H<sub>2</sub>
<italic>sn-1</italic>
, two diastereoisomers); 60.5–60.4
(2s, C<sub>6</sub>
+ C<sub>6′</sub>
); 31.3 (s, <italic>C</italic>
H<sub>2</sub>
fatty alkyl chain); 29.2 (s, <italic>C</italic>
H<sub>2</sub>
fatty alkyl chain); 29.0 (s, <italic>C</italic>
H<sub>2</sub>
fatty alkyl chain); 28.8 (s, <italic>C</italic>
H<sub>2</sub>
fatty
alkyl chain); 28.7 (s, <italic>C</italic>
H<sub>2</sub>
fatty alkyl
chain); 25.6 (s, <italic>C</italic>
H<sub>2</sub>
fatty alkyl chain);
22.1 (s, <italic>C</italic>
H<sub>2</sub>
fatty alkyl chain); 13.9
(s, <italic>C</italic>
H<sub>3</sub>
fatty alkyl chain).</p>
</sec>
</sec>
<sec id="sec5-2"><label>5.2</label>
<title>Liposome Preparation</title>
<p>Appropriate
amounts of lipids (ca. 10 mg) and unlabeled OHM (0.56 mg for lipid-to-OHM
molar ratio, Ri, of 15 and 0.28 mg for Ri = 30) were weighted and
cosolubilized in an organic solvent mixture [chloroform/methanol (2:1)]
to ensure a complete mixing of the components, followed by solvent
evaporation under a nitrogen gas flow, removal of solvent traces using
a high-speed vacuum apparatus, and suspending in water and overnight
freeze-drying. Deuterium-depleted water (100 μL) was added to
obtain lipid hydration, <italic>h</italic>
= 90% [<italic>h</italic>
= mass of water over the total mass of the system (phospholipids
and water)]. After shaking in a vortex mixer, the last step was carried
out, which consisted of three freeze–thaw cycles; samples were
frozen in liquid nitrogen for 30 s, heated at 45 °C for 10 min
in a water bath, and shaken again for better sample homogeneity. Three
different systems of lipids with and without OHM were prepared following
this protocol: one with pure POPC-<sup>2</sup>
H<sub>31</sub>
(PC,
purchased from Avanti Polar Lipids) and two others containing PC,
CHOL, and egg SGML. The molar ratios were the following for PC/SGML/CHOL:
10/60/30 and 10/85/05 (mw<sub>POPC-<sup>2</sup>
H<sub>31</sub>
</sub>
= 760.08 g·mol<sup>–1</sup>
; mw<sub>SGML</sub>
= 710.96 g·mol<sup>–1</sup>
; mw<sub>CHOL</sub>
= 386.65
g·mol<sup>–1</sup>
; mw<sub>OHM</sub>
= 654 g·mol<sup>–1</sup>
; and mw<sub>H<sub>2</sub>
O</sub>
= 18.01 g·mol<sup>–1</sup>
). Samples with deuterated OHM and protonated lipids
were prepared using the same procedure.</p>
</sec>
<sec id="sec5-3"><label>5.3</label>
<title>Solid-State
and Liquid-State NMR</title>
<p>NMR experiments were carried out on
Bruker Avance III 800 MHz (18.8
T) and 400 MHz (9.4 T) spectrometers. Wide-line <sup>2</sup>
H NMR
spectra were acquired at 122.8 MHz (18.8 T) by means of a quadrupolar
echo pulse sequence<sup><xref ref-type="bibr" rid="ref37">37</xref>
</sup>
with a π/2
pulse width of 4.5 μs, an interpulse delay of 40 μs, and
a recycle delay of 2 s. Typically, 3k to 6k scans were recorded depending
on the concentration and the temperature of the sample. The reference
for solid-state deuterium powder patterns was set to 4.7 ppm for <sup>1</sup>
HO<sup>2</sup>
H. A Lorentzian noise filtering of 100–300
Hz was always applied prior to Fourier transformation from the top
of the echo signal. Quadrature detection was used in all cases. Samples
were allowed to equilibrate for at least 20 min for each temperature
before the NMR signal was acquired. Liquid-state <sup>1</sup>
H NMR
spectra (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">Supporting Information</ext-link>
) were recorded
at 400.1 MHz using a simple pulse sequence, with a π/2 pulse
width of 4 μs and a recycle delay of 5 s. Tetramethylsilane
was used for reference. Nonoriented solid-state NMR spectra were recorded
in the time domain (as free induction decays) and then Fourier transformed.
Individual components were built from the experimental estimates of
quadrupolar splittings, isotropic chemical shifts, and individual
line widths (line width is considered to be constant throughout the
pattern). Small variations were allowed to match with sharp experimental
features of the spectra. For perdeuterated chains, weights are not
variable and depend on the number of deuterons per labeled carbon
position; the individual time-dependent signals were then added accordingly,
leading after Fourier transformation of the multicomponent spectrum.
Once the sharp features are well-defined in the simulation (Δν<sub>Q</sub>
), the only adjustable variable which remains is the ellipsoid <italic>c</italic>
/<italic>a</italic>
ratio that is bound to the orientation
dependence of the global solid-state spectrum,<sup><xref ref-type="bibr" rid="ref38">38</xref>
</sup>
<inline-formula id="d30e1681"><inline-graphic xlink:href="ao-2017-00936p_m001.gif"></inline-graphic>
</inline-formula>
, where θ is the orientation of the
bilayer normal with respect to the magnetic field direction and <italic>c</italic>
and <italic>a</italic>
are the ellipsoid axes. <italic>S</italic>
<sup>CD</sup>
order parameters in the bilayer membranes
are proportional to quadrupolar splittings:<sup><xref ref-type="bibr" rid="ref39">39</xref>
,<xref ref-type="bibr" rid="ref40">40</xref>
</sup>
<italic>S</italic>
<sub><italic>k</italic>
</sub>
<sup arrange="stack">CD</sup>
= 4Δν<sub>Q</sub>
<sup><italic>k</italic>
</sup>
/3<italic>A</italic>
<sub>Q</sub>
.</p>
</sec>
<sec id="sec5-4"><label>5.4</label>
<title>MD Simulations</title>
<p>MD simulations of
the system containing the OHM molecules were performed using a newly
developed force field for such molecules and a standard force field
for lipids (see the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">Supporting Information</ext-link>
).</p>
<p>A bilayer system mimicking the lipid environment of the
SK3 channel was simulated with and without the OHM molecules as a
control (<xref rid="tbl3" ref-type="other">Table <xref rid="tbl3" ref-type="other">3</xref>
</xref>
).
The lipid bilayer is made of molecules of DSPC, <italic>N</italic>
-stearoyl-<sc>d</sc>
-<italic>erythro</italic>
-sphingosylphosphorylcholine
(d18:1/18:0 SGML), and CHOL. The lipid composition was taken from
previous work,<sup><xref ref-type="bibr" rid="ref41">41</xref>
</sup>
where a RAFT domain with
a totally saturated lipid, SGML, and CHOL molecules was analyzed.
It is worth to notice that CHOL strongly interacts with saturated
lipids and SGML, and therefore RAFT domains are enriched in saturated
lipids.<sup><xref ref-type="bibr" rid="ref42">42</xref>
,<xref ref-type="bibr" rid="ref43">43</xref>
</sup>
</p>
<table-wrap id="tbl3" position="float"><label>Table 3</label>
<caption><title>Composition of the
Simulated Systems</title>
</caption>
<table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col>
<col align="left"></col>
<col align="left"></col>
<col align="left"></col>
</colgroup>
<thead><tr><th style="border:none;" align="center"> </th>
<th style="border:none;" align="center">number of lipids (fractional composition)</th>
<th style="border:none;" align="center">water molecules</th>
<th style="border:none;" align="center">simulated
time (ns)</th>
</tr>
</thead>
<tbody><tr><td style="border:none;" align="left">RAFT</td>
<td style="border:none;" align="left">DSPC: 54 (0.36)</td>
<td style="border:none;" align="left">5586</td>
<td style="border:none;" align="left">200</td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">SGML: 44 (0.30)</td>
<td style="border:none;" align="left"> </td>
<td style="border:none;" align="left"> </td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">CHOL: 50 (0.34)</td>
<td style="border:none;" align="left"> </td>
<td style="border:none;" align="left"> </td>
</tr>
<tr><td style="border:none;" align="left">RAFT–OHM</td>
<td style="border:none;" align="left">DSPC: 54 (0.33)</td>
<td style="border:none;" align="left">5901</td>
<td style="border:none;" align="left">400</td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">SGML: 44 (0.27)</td>
<td style="border:none;" align="left"> </td>
<td style="border:none;" align="left"> </td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">CHOL: 50 (0.31)</td>
<td style="border:none;" align="left"> </td>
<td style="border:none;" align="left"> </td>
</tr>
<tr><td style="border:none;" align="left"> </td>
<td style="border:none;" align="left">OHM:
14 (0.09)</td>
<td style="border:none;" align="left"> </td>
<td style="border:none;" align="left"> </td>
</tr>
</tbody>
</table>
</table-wrap>
<p>All MD simulations were performed using the
Gromacs package version
4.5.<sup><xref ref-type="bibr" rid="ref44">44</xref>
</sup>
A direct cutoff for nonbonded interactions
of 1.6 nm and particle mesh Ewald for long-range electrostatics were
applied.<sup><xref ref-type="bibr" rid="ref45">45</xref>
</sup>
Berendsen<sup><xref ref-type="bibr" rid="ref46">46</xref>
</sup>
baths were used to couple the simulation boxes with an
isotropic pressure of 1 atm and to the reference temperature of 310
K. The OHM molecules together with the lipid membrane and water molecules
were coupled to separate the Berendsen thermostats with a relaxation
time of 0.1 ps. All bond lengths were constrained using the LINCS
algorithm,<sup><xref ref-type="bibr" rid="ref47">47</xref>
</sup>
whereas the SETTLE algorithm<sup><xref ref-type="bibr" rid="ref48">48</xref>
</sup>
was used for water molecules. All systems were
solvated with SPC water molecules.<sup><xref ref-type="bibr" rid="ref49">49</xref>
</sup>
The
time step in all simulations was set to 2 fs. The simulation time
for each system is shown in <xref rid="tbl3" ref-type="other">Table <xref rid="tbl3" ref-type="other">3</xref>
</xref>
.</p>
<sec id="sec5-4-1"><label>5.4.1</label>
<title>Starting Conformations</title>
<p>The starting
conformations for all systems were taken from previous publication.<sup><xref ref-type="bibr" rid="ref41">41</xref>
</sup>
It is worth to notice that in that publication,
the systems were simulated using another force field for the lipids,
so the system without the OHM molecules was simulated again using
the previously mentioned conditions. Most properties of this system
were practically identical to those reported previously.<sup><xref ref-type="bibr" rid="ref41">41</xref>
</sup>
</p>
<p>The protocol to insert the OHM molecules
into the membrane was described previously for the insertion of proteins
into membranes.<sup><xref ref-type="bibr" rid="ref50">50</xref>
</sup>
Basically, it consists
of several steps of membrane and OHM molecule preparations, followed
by series of scaling of the lipid position and energy minimization
calculations (see the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">Supporting Information</ext-link>
).</p>
<p>All trajectories were analyzed using the standard tools
from the
Gromacs package and custom tcl/tk scripts in the visual molecular
dynamics environment.<sup><xref ref-type="bibr" rid="ref51">51</xref>
</sup>
Different properties
were calculated for each of the simulated systems along the trajectory
(density profiles, RDFs, order parameters, etc.). Temporal averages
were calculated in all cases along the production stage (the last
50 ns of the simulation).</p>
<p>Additionally, density profile and
order parameters of each system
were measured not only to quantify the overall organization of the
lipid component but also to evaluate the production stage of each
simulation. Therefore, a system was considered to be equilibrated
when no further changes were found in those properties on at least
three consecutive intervals of 20 ns.</p>
</sec>
<sec id="sec5-4-2"><label>5.4.2</label>
<title>Analysis</title>
<p>Most of the analysis on
the trajectories was done using standard tools from the Gromacs package
4.5. The number density profile, the order parameter, the RDF, and
the interleaflet mixing were calculated and averaged over the last
20 ns of each simulation. The number of hydrogen bonds was calculated
based on an angle acceptor–donor-Hydrogen cutoff of 30°
and a distance donor–acceptor cutoff of 3.5 Å.</p>
<p>The
area per lipid was calculated from the P atom projection into a 2D
surface using the Voronoi tessellation technique as it was previously
used and described in the literature.<sup><xref ref-type="bibr" rid="ref52">52</xref>
−<xref ref-type="bibr" rid="ref54">54</xref>
</sup>
It should be mentioned
that this approach provides only an approximation to the real values
because it tends to overestimate the area of smaller molecules and
underestimate that of larger ones.<sup><xref ref-type="bibr" rid="ref55">55</xref>
</sup>
Nevertheless,
it allows us to obtain a good comparative estimation of the area per
lipid for each component and its variation when the OHM molecules
are present.</p>
<p>The thickness was measured as the average distance,
in a direction
normal to the membrane, between the centers of mass of the phosphorous
atoms in each of the two layers to minimize the errors due to lateral
fluctuations. The obtained errors are quite small in comparison to
the experimental values because they came from an average value along
the trajectory but are comparable to the experimental measurements
of the thickness using the Luzzati method.<sup><xref ref-type="bibr" rid="ref56">56</xref>
,<xref ref-type="bibr" rid="ref57">57</xref>
</sup>
The order parameters were calculated for the tail C carbon atoms
located on a slice at different depths into the membrane, to check
the OHM-induced variation of the lipid order along the membrane normal.</p>
</sec>
</sec>
</sec>
</body>
<back><notes id="notes1" notes-type="si"><title>Supporting Information Available</title>
<p>The Supporting Information is
available free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications website</ext-link>
at DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acsomega.7b00936">10.1021/acsomega.7b00936</ext-link>
.<list id="silist" list-type="simple"><list-item><p>Synthesis of deuterated
OHM at the <italic>sn</italic>
2 position; NMR spectra of the intermediates
and CD3-OHM in solution;
and MD starting conformation and parameter development (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00936/suppl_file/ao7b00936_si_001.pdf">PDF</ext-link>
)</p>
</list-item>
</list>
</p>
</notes>
<sec sec-type="supplementary-material"><title>Supplementary Material</title>
<supplementary-material content-type="local-data" id="sifile1"><media xlink:href="ao7b00936_si_001.pdf"><caption><p>ao7b00936_si_001.pdf</p>
</caption>
</media>
</supplementary-material>
</sec>
<notes id="notes2" notes-type="COI-statement"><p>The authors
declare no competing financial interest.</p>
</notes>
<ack><title>Acknowledgments</title>
<p>Financial support
from the TGIR-RMN-THC Fr3050 CNRS
for conducting the research and La Ligue contre le cancer is gratefully
acknowledged. The Aquitaine Government is also thanked for providing
grants to set up the NMR platform. This work was also funded by “Université
François Rabelais de Tours,” “INSERM,”
The Région Centre Val de Loire (LIPIDS Project ARD 2020 Biomédicaments),
the committee Indre et Loire of “La Ligue Contre le Cancer,”
the association CANCEN and Tours’ hospital oncology association
ACORT and by Agencia Nacional de Promoción Científica
y Tecnológica, project PICT 2014 - 1249.</p>
</ack>
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