TRPV5 is a six-transmembrane domain channel that is selective to Ca²⁺ ions. To study the topology of the selectivity filter, cysteine mutants at positions 541 to 547 were studied as heterotetramers using dimeric constructs that couple in a tandem the control channel with a cysteine-bearing subunit. Whole-cell currents of dimeric constructs D542C, G543C, P544C, A545C, and Y547C were rapidly inhibited by positively charged MTSMT, MTSEA, and MTSET reagents whereas D542C, P544C, and A545C were inhibited by the negatively charged MTSES. In contrast, I541C dimer remained insensitive to positive and negative reagents. However the I541C / D542G and the I541C / D542N dimeric constructs were rapidly (< 30 s) and strongly inhibited by positively and negatively charged MTS reagents suggesting that removing two out of the four carboxylate residues at position 542 disrupts a constriction point in the selectivity filter. Altogether 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 3-D structure of the selectivity filter in K⁺channels where the main-chain carbonyls were shown to project toward a narrow permeation pathway. The I541C data further suggest that the selectivity filter of TRPV5 espouses a specific conformation that restrains channel accessibility in the presence of four carboxylate residues at position 542.