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 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 (