Topology of the selectivity filter of a TRPV channel: rapid accessibility of contiguous residues from the external medium
Département de Physiologie and the Membrane Protein Research Group, Université de Montréal, Montréal, Québec, Canada Submitted 5 September 2007 ; accepted in final form 11 October 2007 The transient receptor potential type V5 (TRPV5) channel is a six-transmembrane domain ion channel that is highly s...
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| container_title | American Journal of Physiology: Cell Physiology |
| container_volume | 293 |
| creator | Dodier, Yolaine Dionne, Francois Raybaud, Alexandra Sauve, Remy Parent, Lucie |
| description | Département de Physiologie and the Membrane Protein Research Group, Université de Montréal, Montréal, Québec, Canada
Submitted 5 September 2007
; accepted in final form 11 October 2007
The transient receptor potential type V5 (TRPV5) channel is a six-transmembrane domain ion channel that is highly selective to Ca 2+ . To study the topology of the selectivity filter using the substituted cysteine accessibility method (SCAM), cysteine mutants at positions 541–547 were studied as heterotetramers using dimeric constructs that couple the control channel in tandem with a cysteine-bearing subunit. Whole cell currents of dimeric constructs D542C, G543C, P544C, A545C, and Y547C were rapidly inhibited by positively charged 2-(trimethyl ammonium)methyl methane thiosulfonate bromide (MTSMT), 2-(aminoethyl)methane thiosulfonate bromide (MTSEA), and 2-(trimethyl ammonium)ethyl methane thiosulfonate bromide (MTSET) reagents, whereas D542C, P544C, and A545C were inhibited only by negatively charged sodium 2-(sulfonatoethyl)methane thiosulfonate (MTSES). In contrast, the I541C dimer remained insensitive to positive and negative reagents. However, I541C/D542G and I541C/D542N dimeric constructs were rapidly ( |
| doi_str_mv | 10.1152/ajpcell.00406.2007 |
| format | Article |
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Submitted 5 September 2007
; accepted in final form 11 October 2007
The transient receptor potential type V5 (TRPV5) channel is a six-transmembrane domain ion channel that is highly selective to Ca 2+ . To study the topology of the selectivity filter using the substituted cysteine accessibility method (SCAM), cysteine mutants at positions 541–547 were studied as heterotetramers using dimeric constructs that couple the control channel in tandem with a cysteine-bearing subunit. Whole cell currents of dimeric constructs D542C, G543C, P544C, A545C, and Y547C were rapidly inhibited by positively charged 2-(trimethyl ammonium)methyl methane thiosulfonate bromide (MTSMT), 2-(aminoethyl)methane thiosulfonate bromide (MTSEA), and 2-(trimethyl ammonium)ethyl methane thiosulfonate bromide (MTSET) reagents, whereas D542C, P544C, and A545C were inhibited only by negatively charged sodium 2-(sulfonatoethyl)methane thiosulfonate (MTSES). In contrast, the I541C dimer remained insensitive to positive and negative reagents. However, I541C/D542G and I541C/D542N dimeric constructs were rapidly (<30 s) and strongly inhibited by positively and negatively charged methane thiosulfonate reagents, suggesting that removing two of the four carboxylate residues at position 542 disrupts a constriction point in the selectivity filter. Taken together, these results establish that the side chains of contiguous amino acids in the selectivity filter of TRPV5 are rapidly accessible from the external medium, in contrast to the three-dimensional structure of the selectivity filter in K + channels, where main chain carbonyls were shown to project toward a narrow permeation pathway. The I541C data further suggest that the selectivity filter of the TRPV5 channel espouses a specific conformation that restrains accessibility in the presence of four carboxylate residues at position 542.
calcium; kidney; transport; cysteine; site-directed mutagenesis; electrophysiology; methane thiosulfonate reagents; three-dimensional homology modeling; ion channel; transient receptor potential
Address for reprint requests and other correspondence: L. Parent, Dept. of Physiology, Université de Montréal, PO Box 6128, Downtown Station, Montréal, QC, Canada H3C 3J7 (e-mail: lucie.parent{at}umontreal.ca )</description><identifier>ISSN: 0363-6143</identifier><identifier>EISSN: 1522-1563</identifier><identifier>DOI: 10.1152/ajpcell.00406.2007</identifier><identifier>PMID: 17942632</identifier><identifier>CODEN: AJPCDD</identifier><language>eng</language><publisher>United States: American Physiological Society</publisher><subject>Amino Acid Sequence ; Amino acids ; Animals ; Aspartic Acid - metabolism ; Binding Sites - drug effects ; Calcium ; Calcium - metabolism ; Cell Membrane - metabolism ; Cells ; Extracellular Space - metabolism ; Female ; Indicators and Reagents - pharmacology ; Ions ; Isoleucine - metabolism ; Membranes ; Mesylates - pharmacology ; Mutation ; Protein Conformation ; Rabbits ; Studies ; TRPV Cation Channels - chemistry ; TRPV Cation Channels - drug effects ; TRPV Cation Channels - metabolism ; Xenopus laevis</subject><ispartof>American Journal of Physiology: Cell Physiology, 2007-12, Vol.293 (6), p.C1962-C1970</ispartof><rights>Copyright American Physiological Society Dec 2007</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c449t-82eaec7472b47a418b3a06b5527ae40bde9c80e370c225e7838cfe4bf7ad935c3</citedby><cites>FETCH-LOGICAL-c449t-82eaec7472b47a418b3a06b5527ae40bde9c80e370c225e7838cfe4bf7ad935c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,3039,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17942632$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Dodier, Yolaine</creatorcontrib><creatorcontrib>Dionne, Francois</creatorcontrib><creatorcontrib>Raybaud, Alexandra</creatorcontrib><creatorcontrib>Sauve, Remy</creatorcontrib><creatorcontrib>Parent, Lucie</creatorcontrib><title>Topology of the selectivity filter of a TRPV channel: rapid accessibility of contiguous residues from the external medium</title><title>American Journal of Physiology: Cell Physiology</title><addtitle>Am J Physiol Cell Physiol</addtitle><description>Département de Physiologie and the Membrane Protein Research Group, Université de Montréal, Montréal, Québec, Canada
Submitted 5 September 2007
; accepted in final form 11 October 2007
The transient receptor potential type V5 (TRPV5) channel is a six-transmembrane domain ion channel that is highly selective to Ca 2+ . To study the topology of the selectivity filter using the substituted cysteine accessibility method (SCAM), cysteine mutants at positions 541–547 were studied as heterotetramers using dimeric constructs that couple the control channel in tandem with a cysteine-bearing subunit. Whole cell currents of dimeric constructs D542C, G543C, P544C, A545C, and Y547C were rapidly inhibited by positively charged 2-(trimethyl ammonium)methyl methane thiosulfonate bromide (MTSMT), 2-(aminoethyl)methane thiosulfonate bromide (MTSEA), and 2-(trimethyl ammonium)ethyl methane thiosulfonate bromide (MTSET) reagents, whereas D542C, P544C, and A545C were inhibited only by negatively charged sodium 2-(sulfonatoethyl)methane thiosulfonate (MTSES). In contrast, the I541C dimer remained insensitive to positive and negative reagents. However, I541C/D542G and I541C/D542N dimeric constructs were rapidly (<30 s) and strongly inhibited by positively and negatively charged methane thiosulfonate reagents, suggesting that removing two of the four carboxylate residues at position 542 disrupts a constriction point in the selectivity filter. Taken together, these results establish that the side chains of contiguous amino acids in the selectivity filter of TRPV5 are rapidly accessible from the external medium, in contrast to the three-dimensional structure of the selectivity filter in K + channels, where main chain carbonyls were shown to project toward a narrow permeation pathway. The I541C data further suggest that the selectivity filter of the TRPV5 channel espouses a specific conformation that restrains accessibility in the presence of four carboxylate residues at position 542.
calcium; kidney; transport; cysteine; site-directed mutagenesis; electrophysiology; methane thiosulfonate reagents; three-dimensional homology modeling; ion channel; transient receptor potential
Address for reprint requests and other correspondence: L. Parent, Dept. of Physiology, Université de Montréal, PO Box 6128, Downtown Station, Montréal, QC, Canada H3C 3J7 (e-mail: lucie.parent{at}umontreal.ca )</description><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Animals</subject><subject>Aspartic Acid - metabolism</subject><subject>Binding Sites - drug effects</subject><subject>Calcium</subject><subject>Calcium - metabolism</subject><subject>Cell Membrane - metabolism</subject><subject>Cells</subject><subject>Extracellular Space - metabolism</subject><subject>Female</subject><subject>Indicators and Reagents - pharmacology</subject><subject>Ions</subject><subject>Isoleucine - metabolism</subject><subject>Membranes</subject><subject>Mesylates - pharmacology</subject><subject>Mutation</subject><subject>Protein Conformation</subject><subject>Rabbits</subject><subject>Studies</subject><subject>TRPV Cation Channels - chemistry</subject><subject>TRPV Cation Channels - drug effects</subject><subject>TRPV Cation Channels - metabolism</subject><subject>Xenopus laevis</subject><issn>0363-6143</issn><issn>1522-1563</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkV-L1DAUxYso7rj6BXyQ4INvHfM_7b7J4KqwoMjoa0jT22mGtKlNq_bbm87MogjiUwL3dw73npNlzwneEiLoa3McLHi_xZhjuaUYqwfZJg1oToRkD7MNZpLlknB2lT2J8YgTSGX5OLsiqkw_RjfZsg9D8OGwoNCgqQUUwYOd3Hc3LahxfoJxnRi0__zpK7Kt6XvwN2g0g6uRsRZidJXzK50wG_rJHeYwRzRCdPUMETVj6E7O8DOZ9cajDmo3d0-zR43xEZ5d3uvsy-3b_e59fvfx3Yfdm7vccl5OeUHBgFVc0Yorw0lRMYNlJQRVBjiuaihtgYEpbCkVoApW2AZ41ShTl0xYdp29OvsOY_iWFpp05-Kam-khLapliYXEJf4vSLFQgmORwJd_gccwr5clhmHGCC1IgugZsmOIcYRGD6PrzLhogvVan77Up0_16bW-JHpxcZ6rlNJvyaWvBNycgdYd2h9uBD20S3SnBvXt7P0-pXzvTEumpd6RUlI91E0Sb_8tvt_mDxH7BbiOvl4</recordid><startdate>20071201</startdate><enddate>20071201</enddate><creator>Dodier, Yolaine</creator><creator>Dionne, Francois</creator><creator>Raybaud, Alexandra</creator><creator>Sauve, Remy</creator><creator>Parent, Lucie</creator><general>American Physiological Society</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>7QP</scope><scope>7TS</scope><scope>7X8</scope></search><sort><creationdate>20071201</creationdate><title>Topology of the selectivity filter of a TRPV channel: rapid accessibility of contiguous residues from the external medium</title><author>Dodier, Yolaine ; Dionne, Francois ; Raybaud, Alexandra ; Sauve, Remy ; Parent, Lucie</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c449t-82eaec7472b47a418b3a06b5527ae40bde9c80e370c225e7838cfe4bf7ad935c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Amino Acid Sequence</topic><topic>Amino acids</topic><topic>Animals</topic><topic>Aspartic Acid - metabolism</topic><topic>Binding Sites - drug effects</topic><topic>Calcium</topic><topic>Calcium - metabolism</topic><topic>Cell Membrane - metabolism</topic><topic>Cells</topic><topic>Extracellular Space - metabolism</topic><topic>Female</topic><topic>Indicators and Reagents - pharmacology</topic><topic>Ions</topic><topic>Isoleucine - metabolism</topic><topic>Membranes</topic><topic>Mesylates - pharmacology</topic><topic>Mutation</topic><topic>Protein Conformation</topic><topic>Rabbits</topic><topic>Studies</topic><topic>TRPV Cation Channels - chemistry</topic><topic>TRPV Cation Channels - drug effects</topic><topic>TRPV Cation Channels - metabolism</topic><topic>Xenopus laevis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Dodier, Yolaine</creatorcontrib><creatorcontrib>Dionne, Francois</creatorcontrib><creatorcontrib>Raybaud, Alexandra</creatorcontrib><creatorcontrib>Sauve, Remy</creatorcontrib><creatorcontrib>Parent, Lucie</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Physical Education Index</collection><collection>MEDLINE - Academic</collection><jtitle>American Journal of Physiology: Cell Physiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Dodier, Yolaine</au><au>Dionne, Francois</au><au>Raybaud, Alexandra</au><au>Sauve, Remy</au><au>Parent, Lucie</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Topology of the selectivity filter of a TRPV channel: rapid accessibility of contiguous residues from the external medium</atitle><jtitle>American Journal of Physiology: Cell Physiology</jtitle><addtitle>Am J Physiol Cell Physiol</addtitle><date>2007-12-01</date><risdate>2007</risdate><volume>293</volume><issue>6</issue><spage>C1962</spage><epage>C1970</epage><pages>C1962-C1970</pages><issn>0363-6143</issn><eissn>1522-1563</eissn><coden>AJPCDD</coden><abstract>Département de Physiologie and the Membrane Protein Research Group, Université de Montréal, Montréal, Québec, Canada
Submitted 5 September 2007
; accepted in final form 11 October 2007
The transient receptor potential type V5 (TRPV5) channel is a six-transmembrane domain ion channel that is highly selective to Ca 2+ . To study the topology of the selectivity filter using the substituted cysteine accessibility method (SCAM), cysteine mutants at positions 541–547 were studied as heterotetramers using dimeric constructs that couple the control channel in tandem with a cysteine-bearing subunit. Whole cell currents of dimeric constructs D542C, G543C, P544C, A545C, and Y547C were rapidly inhibited by positively charged 2-(trimethyl ammonium)methyl methane thiosulfonate bromide (MTSMT), 2-(aminoethyl)methane thiosulfonate bromide (MTSEA), and 2-(trimethyl ammonium)ethyl methane thiosulfonate bromide (MTSET) reagents, whereas D542C, P544C, and A545C were inhibited only by negatively charged sodium 2-(sulfonatoethyl)methane thiosulfonate (MTSES). In contrast, the I541C dimer remained insensitive to positive and negative reagents. However, I541C/D542G and I541C/D542N dimeric constructs were rapidly (<30 s) and strongly inhibited by positively and negatively charged methane thiosulfonate reagents, suggesting that removing two of the four carboxylate residues at position 542 disrupts a constriction point in the selectivity filter. Taken together, these results establish that the side chains of contiguous amino acids in the selectivity filter of TRPV5 are rapidly accessible from the external medium, in contrast to the three-dimensional structure of the selectivity filter in K + channels, where main chain carbonyls were shown to project toward a narrow permeation pathway. The I541C data further suggest that the selectivity filter of the TRPV5 channel espouses a specific conformation that restrains accessibility in the presence of four carboxylate residues at position 542.
calcium; kidney; transport; cysteine; site-directed mutagenesis; electrophysiology; methane thiosulfonate reagents; three-dimensional homology modeling; ion channel; transient receptor potential
Address for reprint requests and other correspondence: L. Parent, Dept. of Physiology, Université de Montréal, PO Box 6128, Downtown Station, Montréal, QC, Canada H3C 3J7 (e-mail: lucie.parent{at}umontreal.ca )</abstract><cop>United States</cop><pub>American Physiological Society</pub><pmid>17942632</pmid><doi>10.1152/ajpcell.00406.2007</doi></addata></record> |
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| source | MEDLINE; American Physiological Society; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals |
| subjects | Amino Acid Sequence Amino acids Animals Aspartic Acid - metabolism Binding Sites - drug effects Calcium Calcium - metabolism Cell Membrane - metabolism Cells Extracellular Space - metabolism Female Indicators and Reagents - pharmacology Ions Isoleucine - metabolism Membranes Mesylates - pharmacology Mutation Protein Conformation Rabbits Studies TRPV Cation Channels - chemistry TRPV Cation Channels - drug effects TRPV Cation Channels - metabolism Xenopus laevis |
| title | Topology of the selectivity filter of a TRPV channel: rapid accessibility of contiguous residues from the external medium |
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