Different Isoforms of Synapse-associated Protein, SAP97, Are Expressed in the Heart and Have Distinct Effects on the Voltage-gated K+ Channel Kv1.5
The SAP97 isoforms differ by alternatively spliced insertion domains that regulate protein localization and oligomerization. We used reverse transcription-PCR to identify SAP97 isoforms of human and rat myocardium. In Chinese hamster ovary cells, cloned protein expression was studied using Western b...
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Veröffentlicht in: | The Journal of biological chemistry 2003-11, Vol.278 (47), p.47046-47052 |
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description | The SAP97 isoforms differ by alternatively spliced insertion domains that regulate protein localization and oligomerization. We used reverse transcription-PCR to identify SAP97 isoforms of human and rat myocardium. In Chinese hamster ovary cells, cloned protein expression was studied using Western blot, confocal imaging of green fluorescent protein-tagged proteins, and patch clamp technique. The two main cardiac SAP97 isoforms contained both I3 and I1B inserts and differed by the I1A insert. Both isoforms co-precipitated with hKv1.5 channels. Only the isoform lacking I1A increased the current (by 215 ± 22%), whatever the level of channel expression. To examine the involvement of the proline-rich I1A insert in the effect of SAP97, a W623F mutation in the Src homology 3 domain was created, and that restored the effect of the SAP97 on current. SAP97 isoform containing an I1A and I2 domain instead of the I3 domain stimulated the current, whereas SAP97 after deletion of the Src homology 3 and guanylate kinase-like domains did not. In cells co-expressing I3(+I1A) or I3(-I1A), green fluorescent protein-tagged Kv1.5 channels were organized in plaque-like structures at the plasma membrane level, whereas intracellular aggregates of channels predominated with the I2 isoform. The two cardiac SAP97 isoforms have different effects on the hKv1.5 current, depending on their capacity to form channel clusters. |
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We used reverse transcription-PCR to identify SAP97 isoforms of human and rat myocardium. In Chinese hamster ovary cells, cloned protein expression was studied using Western blot, confocal imaging of green fluorescent protein-tagged proteins, and patch clamp technique. The two main cardiac SAP97 isoforms contained both I3 and I1B inserts and differed by the I1A insert. Both isoforms co-precipitated with hKv1.5 channels. Only the isoform lacking I1A increased the current (by 215 ± 22%), whatever the level of channel expression. To examine the involvement of the proline-rich I1A insert in the effect of SAP97, a W623F mutation in the Src homology 3 domain was created, and that restored the effect of the SAP97 on current. SAP97 isoform containing an I1A and I2 domain instead of the I3 domain stimulated the current, whereas SAP97 after deletion of the Src homology 3 and guanylate kinase-like domains did not. In cells co-expressing I3(+I1A) or I3(-I1A), green fluorescent protein-tagged Kv1.5 channels were organized in plaque-like structures at the plasma membrane level, whereas intracellular aggregates of channels predominated with the I2 isoform. The two cardiac SAP97 isoforms have different effects on the hKv1.5 current, depending on their capacity to form channel clusters.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M308463200</identifier><identifier>PMID: 12970345</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Adaptor Proteins, Signal Transducing ; Animals ; Cloning, Molecular ; Discs Large Homolog 1 Protein ; Electrophysiology ; Heart Atria - chemistry ; Humans ; Kv1.5 Potassium Channel ; Membrane Proteins ; Myocardium - chemistry ; Myocardium - metabolism ; Nerve Tissue Proteins - analysis ; Nerve Tissue Proteins - biosynthesis ; Nerve Tissue Proteins - physiology ; Potassium Channels, Voltage-Gated - chemistry ; Potassium Channels, Voltage-Gated - metabolism ; Protein Binding ; Protein Isoforms - analysis ; Protein Isoforms - biosynthesis ; Protein Isoforms - metabolism ; Protein Isoforms - physiology ; Rats ; Rats, Wistar ; Transfection</subject><ispartof>The Journal of biological chemistry, 2003-11, Vol.278 (47), p.47046-47052</ispartof><rights>2003 © 2003 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c475t-70de9dc7f2d4f71296eb9a785684d754572ae4cdb5a276b6763d45a773995ce13</citedby><cites>FETCH-LOGICAL-c475t-70de9dc7f2d4f71296eb9a785684d754572ae4cdb5a276b6763d45a773995ce13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12970345$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Godreau, David</creatorcontrib><creatorcontrib>Vranckx, Roger</creatorcontrib><creatorcontrib>Maguy, Ange</creatorcontrib><creatorcontrib>Goyenvalle, Catherine</creatorcontrib><creatorcontrib>Hatem, Stéphane N.</creatorcontrib><title>Different Isoforms of Synapse-associated Protein, SAP97, Are Expressed in the Heart and Have Distinct Effects on the Voltage-gated K+ Channel Kv1.5</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The SAP97 isoforms differ by alternatively spliced insertion domains that regulate protein localization and oligomerization. We used reverse transcription-PCR to identify SAP97 isoforms of human and rat myocardium. In Chinese hamster ovary cells, cloned protein expression was studied using Western blot, confocal imaging of green fluorescent protein-tagged proteins, and patch clamp technique. The two main cardiac SAP97 isoforms contained both I3 and I1B inserts and differed by the I1A insert. Both isoforms co-precipitated with hKv1.5 channels. Only the isoform lacking I1A increased the current (by 215 ± 22%), whatever the level of channel expression. To examine the involvement of the proline-rich I1A insert in the effect of SAP97, a W623F mutation in the Src homology 3 domain was created, and that restored the effect of the SAP97 on current. SAP97 isoform containing an I1A and I2 domain instead of the I3 domain stimulated the current, whereas SAP97 after deletion of the Src homology 3 and guanylate kinase-like domains did not. In cells co-expressing I3(+I1A) or I3(-I1A), green fluorescent protein-tagged Kv1.5 channels were organized in plaque-like structures at the plasma membrane level, whereas intracellular aggregates of channels predominated with the I2 isoform. The two cardiac SAP97 isoforms have different effects on the hKv1.5 current, depending on their capacity to form channel clusters.</description><subject>Adaptor Proteins, Signal Transducing</subject><subject>Animals</subject><subject>Cloning, Molecular</subject><subject>Discs Large Homolog 1 Protein</subject><subject>Electrophysiology</subject><subject>Heart Atria - chemistry</subject><subject>Humans</subject><subject>Kv1.5 Potassium Channel</subject><subject>Membrane Proteins</subject><subject>Myocardium - chemistry</subject><subject>Myocardium - metabolism</subject><subject>Nerve Tissue Proteins - analysis</subject><subject>Nerve Tissue Proteins - biosynthesis</subject><subject>Nerve Tissue Proteins - physiology</subject><subject>Potassium Channels, Voltage-Gated - chemistry</subject><subject>Potassium Channels, Voltage-Gated - metabolism</subject><subject>Protein Binding</subject><subject>Protein Isoforms - analysis</subject><subject>Protein Isoforms - biosynthesis</subject><subject>Protein Isoforms - metabolism</subject><subject>Protein Isoforms - physiology</subject><subject>Rats</subject><subject>Rats, Wistar</subject><subject>Transfection</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kUGPEyEUx4nRuLV69Wg4GC_uVJiBYebYdKvd7Bo3WTXeCANvOmym0AVa3c_hFxadJnuSkHDg934P3h-h15QsKBHsw12nF58r0rC6Kgl5gmaUNFVRcfrjKZoRUtKiLXlzhl7EeEfyYi19js5o2QpSMT5Dvy9s30MAl_Bl9L0Pu4h9j28fnNpHKFSMXluVwOCb4BNYd45vlzetOMfLAHj9ax8gxnxrHU4D4A2okLByBm_UEfCFjck6nfA6N9Epqyfsux-T2kKx_We-eo9Xg3IORnx1pAv-Ej3r1Rjh1emco28f119Xm-L6y6fL1fK60EzwVAhioDVa9KVhvchfqqFrlWh43TAjOOOiVMC06bgqRd3Voq4M40qIqm25BlrN0bvJuw_-_gAxyZ2NGsZROfCHKAWtmrLibQYXE6iDjzFAL_fB7lR4kJTIvzHIHIN8jCEXvDmZD90OzCN-mnsG3k7AYLfDTxtAdtbrAXayFI1kIm-SXXPUTBjkMRwtBBm1BafB5BKdpPH2f0_4AwdyoNg</recordid><startdate>20031121</startdate><enddate>20031121</enddate><creator>Godreau, David</creator><creator>Vranckx, Roger</creator><creator>Maguy, Ange</creator><creator>Goyenvalle, Catherine</creator><creator>Hatem, Stéphane N.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20031121</creationdate><title>Different Isoforms of Synapse-associated Protein, SAP97, Are Expressed in the Heart and Have Distinct Effects on the Voltage-gated K+ Channel Kv1.5</title><author>Godreau, David ; Vranckx, Roger ; Maguy, Ange ; Goyenvalle, Catherine ; Hatem, Stéphane N.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c475t-70de9dc7f2d4f71296eb9a785684d754572ae4cdb5a276b6763d45a773995ce13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Adaptor Proteins, Signal Transducing</topic><topic>Animals</topic><topic>Cloning, Molecular</topic><topic>Discs Large Homolog 1 Protein</topic><topic>Electrophysiology</topic><topic>Heart Atria - chemistry</topic><topic>Humans</topic><topic>Kv1.5 Potassium Channel</topic><topic>Membrane Proteins</topic><topic>Myocardium - chemistry</topic><topic>Myocardium - metabolism</topic><topic>Nerve Tissue Proteins - analysis</topic><topic>Nerve Tissue Proteins - biosynthesis</topic><topic>Nerve Tissue Proteins - physiology</topic><topic>Potassium Channels, Voltage-Gated - chemistry</topic><topic>Potassium Channels, Voltage-Gated - metabolism</topic><topic>Protein Binding</topic><topic>Protein Isoforms - analysis</topic><topic>Protein Isoforms - biosynthesis</topic><topic>Protein Isoforms - metabolism</topic><topic>Protein Isoforms - physiology</topic><topic>Rats</topic><topic>Rats, Wistar</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Godreau, David</creatorcontrib><creatorcontrib>Vranckx, Roger</creatorcontrib><creatorcontrib>Maguy, Ange</creatorcontrib><creatorcontrib>Goyenvalle, Catherine</creatorcontrib><creatorcontrib>Hatem, Stéphane N.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Godreau, David</au><au>Vranckx, Roger</au><au>Maguy, Ange</au><au>Goyenvalle, Catherine</au><au>Hatem, Stéphane N.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Different Isoforms of Synapse-associated Protein, SAP97, Are Expressed in the Heart and Have Distinct Effects on the Voltage-gated K+ Channel Kv1.5</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2003-11-21</date><risdate>2003</risdate><volume>278</volume><issue>47</issue><spage>47046</spage><epage>47052</epage><pages>47046-47052</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>The SAP97 isoforms differ by alternatively spliced insertion domains that regulate protein localization and oligomerization. We used reverse transcription-PCR to identify SAP97 isoforms of human and rat myocardium. In Chinese hamster ovary cells, cloned protein expression was studied using Western blot, confocal imaging of green fluorescent protein-tagged proteins, and patch clamp technique. The two main cardiac SAP97 isoforms contained both I3 and I1B inserts and differed by the I1A insert. Both isoforms co-precipitated with hKv1.5 channels. Only the isoform lacking I1A increased the current (by 215 ± 22%), whatever the level of channel expression. To examine the involvement of the proline-rich I1A insert in the effect of SAP97, a W623F mutation in the Src homology 3 domain was created, and that restored the effect of the SAP97 on current. SAP97 isoform containing an I1A and I2 domain instead of the I3 domain stimulated the current, whereas SAP97 after deletion of the Src homology 3 and guanylate kinase-like domains did not. In cells co-expressing I3(+I1A) or I3(-I1A), green fluorescent protein-tagged Kv1.5 channels were organized in plaque-like structures at the plasma membrane level, whereas intracellular aggregates of channels predominated with the I2 isoform. The two cardiac SAP97 isoforms have different effects on the hKv1.5 current, depending on their capacity to form channel clusters.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>12970345</pmid><doi>10.1074/jbc.M308463200</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Adaptor Proteins, Signal Transducing Animals Cloning, Molecular Discs Large Homolog 1 Protein Electrophysiology Heart Atria - chemistry Humans Kv1.5 Potassium Channel Membrane Proteins Myocardium - chemistry Myocardium - metabolism Nerve Tissue Proteins - analysis Nerve Tissue Proteins - biosynthesis Nerve Tissue Proteins - physiology Potassium Channels, Voltage-Gated - chemistry Potassium Channels, Voltage-Gated - metabolism Protein Binding Protein Isoforms - analysis Protein Isoforms - biosynthesis Protein Isoforms - metabolism Protein Isoforms - physiology Rats Rats, Wistar Transfection |
title | Different Isoforms of Synapse-associated Protein, SAP97, Are Expressed in the Heart and Have Distinct Effects on the Voltage-gated K+ Channel Kv1.5 |
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