title: Topology of the selectivity filter of a TRPV channel: rapid accessibility of contiguous residues from the external medium.
abstract: 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.
authors.affiliation: Department of Physiology and the Membrane Protein Research Group, Université de Montréal, Montréal, QC, Canada.
authors.forename: Yolaine
authors.initials: Y
authors.lastname: Dodier
authors.forename: François
authors.initials: F
authors.lastname: Dionne
authors.forename: Alexandra
authors.initials: A
authors.lastname: Raybaud
authors.forename: Rémy
authors.initials: R
authors.lastname: Sauvé
authors.forename: Lucie
authors.initials: L
authors.lastname: Parent
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D007202:Indicators and Reagents / Q000494:pharmacology; D007532:Isoleucine / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D050916:TRPV Cation Channels / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D007202:Indicators and Reagents; D008698:Mesylates; D050916:TRPV Cation Channels; D007532:Isoleucine; D001224:Aspartic Acid; C074502:methanethiosulfonate; D002118:Calcium