Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels
We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a r...
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| creator | Leung, Yuk M Kang, Youhou Xia, Fuzhen Sheu, Laura Gao, Xiaodong Xie, Huanli Tsushima, Robert G Gaisano, Herbert Y |
| description | We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P |
| doi_str_mv | 10.1042/BJ20041625 |
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| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_1134947</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>67529710</sourcerecordid><originalsourceid>FETCH-LOGICAL-c376t-6b2d6a1fb8798f276b5fa5ae276415ae14d9ce026df56502fd0cf5af8a6d17ff3</originalsourceid><addsrcrecordid>eNpVkUlPwzAQhS0EgrJc-AHIJw5IAY_rJb0gAWJH6gXOxklsapTYwU6B_nuMWrGcZqT55s0bPYT2gRwDYfTk_I4SwkBQvoZGwCQpSknLdTQiVLBCEApbaDulV0KAEUY20RZwDiUv5Qg9T3vjsQ2xw8HitPCD_nS-gDPsEta4C9HgPgzGD9j5mavcECIeZtrjD9c2xbDozb-tLHL_To8B15nxpk27aMPqNpm9Vd1BT1eXjxc3xcP0-vbi7KGox1IMhahoIzTYqpST0lIpKm411yZ3DHIF1kxqkx9qLBecUNuQ2nJtSy0akNaOd9DpUrefV51p6uw46lb10XU6LlTQTv2feDdTL-FdAYzZhMkscLgSiOFtbtKgOpdq07bamzBPSkhOJxJIBo-WYB1DStHYnyNA1Hcg6jeQDB_8tfWLrhIYfwGyboaM</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>67529710</pqid></control><display><type>article</type><title>Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels</title><source>MEDLINE</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>PubMed Central</source><source>Alma/SFX Local Collection</source><creator>Leung, Yuk M ; Kang, Youhou ; Xia, Fuzhen ; Sheu, Laura ; Gao, Xiaodong ; Xie, Huanli ; Tsushima, Robert G ; Gaisano, Herbert Y</creator><creatorcontrib>Leung, Yuk M ; Kang, Youhou ; Xia, Fuzhen ; Sheu, Laura ; Gao, Xiaodong ; Xie, Huanli ; Tsushima, Robert G ; Gaisano, Herbert Y</creatorcontrib><description>We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.</description><identifier>ISSN: 0264-6021</identifier><identifier>EISSN: 1470-8728</identifier><identifier>DOI: 10.1042/BJ20041625</identifier><identifier>PMID: 15518587</identifier><language>eng</language><publisher>England: Portland Press Ltd</publisher><subject>Antigens, Surface - biosynthesis ; Antigens, Surface - chemistry ; Antigens, Surface - physiology ; Delayed Rectifier Potassium Channels ; Humans ; Ion Channel Gating - physiology ; Kidney - chemistry ; Kidney - cytology ; Kidney - embryology ; Kidney - metabolism ; Nerve Tissue Proteins - chemistry ; Nerve Tissue Proteins - physiology ; Patch-Clamp Techniques - methods ; Potassium Channels, Voltage-Gated - biosynthesis ; Potassium Channels, Voltage-Gated - genetics ; Potassium Channels, Voltage-Gated - metabolism ; Protein Structure, Quaternary - physiology ; Shab Potassium Channels ; Syntaxin 1 ; Transfection - methods</subject><ispartof>Biochemical journal, 2005-04, Vol.387 (Pt 1), p.195-202</ispartof><rights>The Biochemical Society, London 2005</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c376t-6b2d6a1fb8798f276b5fa5ae276415ae14d9ce026df56502fd0cf5af8a6d17ff3</citedby><cites>FETCH-LOGICAL-c376t-6b2d6a1fb8798f276b5fa5ae276415ae14d9ce026df56502fd0cf5af8a6d17ff3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1134947/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1134947/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15518587$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Leung, Yuk M</creatorcontrib><creatorcontrib>Kang, Youhou</creatorcontrib><creatorcontrib>Xia, Fuzhen</creatorcontrib><creatorcontrib>Sheu, Laura</creatorcontrib><creatorcontrib>Gao, Xiaodong</creatorcontrib><creatorcontrib>Xie, Huanli</creatorcontrib><creatorcontrib>Tsushima, Robert G</creatorcontrib><creatorcontrib>Gaisano, Herbert Y</creatorcontrib><title>Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels</title><title>Biochemical journal</title><addtitle>Biochem J</addtitle><description>We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.</description><subject>Antigens, Surface - biosynthesis</subject><subject>Antigens, Surface - chemistry</subject><subject>Antigens, Surface - physiology</subject><subject>Delayed Rectifier Potassium Channels</subject><subject>Humans</subject><subject>Ion Channel Gating - physiology</subject><subject>Kidney - chemistry</subject><subject>Kidney - cytology</subject><subject>Kidney - embryology</subject><subject>Kidney - metabolism</subject><subject>Nerve Tissue Proteins - chemistry</subject><subject>Nerve Tissue Proteins - physiology</subject><subject>Patch-Clamp Techniques - methods</subject><subject>Potassium Channels, Voltage-Gated - biosynthesis</subject><subject>Potassium Channels, Voltage-Gated - genetics</subject><subject>Potassium Channels, Voltage-Gated - metabolism</subject><subject>Protein Structure, Quaternary - physiology</subject><subject>Shab Potassium Channels</subject><subject>Syntaxin 1</subject><subject>Transfection - methods</subject><issn>0264-6021</issn><issn>1470-8728</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkUlPwzAQhS0EgrJc-AHIJw5IAY_rJb0gAWJH6gXOxklsapTYwU6B_nuMWrGcZqT55s0bPYT2gRwDYfTk_I4SwkBQvoZGwCQpSknLdTQiVLBCEApbaDulV0KAEUY20RZwDiUv5Qg9T3vjsQ2xw8HitPCD_nS-gDPsEta4C9HgPgzGD9j5mavcECIeZtrjD9c2xbDozb-tLHL_To8B15nxpk27aMPqNpm9Vd1BT1eXjxc3xcP0-vbi7KGox1IMhahoIzTYqpST0lIpKm411yZ3DHIF1kxqkx9qLBecUNuQ2nJtSy0akNaOd9DpUrefV51p6uw46lb10XU6LlTQTv2feDdTL-FdAYzZhMkscLgSiOFtbtKgOpdq07bamzBPSkhOJxJIBo-WYB1DStHYnyNA1Hcg6jeQDB_8tfWLrhIYfwGyboaM</recordid><startdate>20050401</startdate><enddate>20050401</enddate><creator>Leung, Yuk M</creator><creator>Kang, Youhou</creator><creator>Xia, Fuzhen</creator><creator>Sheu, Laura</creator><creator>Gao, Xiaodong</creator><creator>Xie, Huanli</creator><creator>Tsushima, Robert G</creator><creator>Gaisano, Herbert Y</creator><general>Portland Press Ltd</general><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><scope>5PM</scope></search><sort><creationdate>20050401</creationdate><title>Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels</title><author>Leung, Yuk M ; Kang, Youhou ; Xia, Fuzhen ; Sheu, Laura ; Gao, Xiaodong ; Xie, Huanli ; Tsushima, Robert G ; Gaisano, Herbert Y</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c376t-6b2d6a1fb8798f276b5fa5ae276415ae14d9ce026df56502fd0cf5af8a6d17ff3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Antigens, Surface - biosynthesis</topic><topic>Antigens, Surface - chemistry</topic><topic>Antigens, Surface - physiology</topic><topic>Delayed Rectifier Potassium Channels</topic><topic>Humans</topic><topic>Ion Channel Gating - physiology</topic><topic>Kidney - chemistry</topic><topic>Kidney - cytology</topic><topic>Kidney - embryology</topic><topic>Kidney - metabolism</topic><topic>Nerve Tissue Proteins - chemistry</topic><topic>Nerve Tissue Proteins - physiology</topic><topic>Patch-Clamp Techniques - methods</topic><topic>Potassium Channels, Voltage-Gated - biosynthesis</topic><topic>Potassium Channels, Voltage-Gated - genetics</topic><topic>Potassium Channels, Voltage-Gated - metabolism</topic><topic>Protein Structure, Quaternary - physiology</topic><topic>Shab Potassium Channels</topic><topic>Syntaxin 1</topic><topic>Transfection - methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Leung, Yuk M</creatorcontrib><creatorcontrib>Kang, Youhou</creatorcontrib><creatorcontrib>Xia, Fuzhen</creatorcontrib><creatorcontrib>Sheu, Laura</creatorcontrib><creatorcontrib>Gao, Xiaodong</creatorcontrib><creatorcontrib>Xie, Huanli</creatorcontrib><creatorcontrib>Tsushima, Robert G</creatorcontrib><creatorcontrib>Gaisano, Herbert Y</creatorcontrib><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><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biochemical journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Leung, Yuk M</au><au>Kang, Youhou</au><au>Xia, Fuzhen</au><au>Sheu, Laura</au><au>Gao, Xiaodong</au><au>Xie, Huanli</au><au>Tsushima, Robert G</au><au>Gaisano, Herbert Y</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels</atitle><jtitle>Biochemical journal</jtitle><addtitle>Biochem J</addtitle><date>2005-04-01</date><risdate>2005</risdate><volume>387</volume><issue>Pt 1</issue><spage>195</spage><epage>202</epage><pages>195-202</pages><issn>0264-6021</issn><eissn>1470-8728</eissn><abstract>We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.</abstract><cop>England</cop><pub>Portland Press Ltd</pub><pmid>15518587</pmid><doi>10.1042/BJ20041625</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Antigens, Surface - biosynthesis Antigens, Surface - chemistry Antigens, Surface - physiology Delayed Rectifier Potassium Channels Humans Ion Channel Gating - physiology Kidney - chemistry Kidney - cytology Kidney - embryology Kidney - metabolism Nerve Tissue Proteins - chemistry Nerve Tissue Proteins - physiology Patch-Clamp Techniques - methods Potassium Channels, Voltage-Gated - biosynthesis Potassium Channels, Voltage-Gated - genetics Potassium Channels, Voltage-Gated - metabolism Protein Structure, Quaternary - physiology Shab Potassium Channels Syntaxin 1 Transfection - methods |
| title | Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels |
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