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 2ϩ . 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 (Ͻ30 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 ϩ 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 TRANSIENT RECEPTOR POTENTIAL (TRP) type V (TRPV) channels belong to the six-transmembrane (6-TM) family of ion channels with both NH 2 -and COOH-termini located intracellularly (18). TRPV5 (ECaC1 and CaT2) and TRPV6 (ECaC2, CaT1, and CaT-L) play key roles in renal Ca 2ϩ reabsorption and intestinal Ca 2ϩ absorption, respectively. They both form a distinctive subgroup within the TRP family, as they show strong inward rectification, exhibit an anomalous mole-fraction effect, are activated by low intracellular (9) and is proposed to form the main binding site for divalent cations within the selectivity filter (9, 14). Assuming that Asp 542 controls the pore diameter, the diameter of the selectivity filter has been estimated to vary from 5.4 Å (23) for TRPV6 to 8.5 Å (25) for TRPV5 at physiological pH. Altogether, these observations suppose that the negatively charged carboxylate groups of Asp 542 /Asp 541 project toward the pore lumen and that the selectivity filter of TRPV5/6 is wider than in K ϩ channels. This structural arrangement would contrast w...

Molecular Mechanisms of TRPV1 Channel Activation

“…Similar observations have been made with the calcium impermeable TRPM4 channel, in which mutations in the putative selectivity filter enabled calcium permeation through the receptor [182]. The outer pores of the TRPV6 and TRPV5 have been probed by using the substituted cysteine accessibility method (SCAM), and the results are also consistent with a pore topology similar to Kv channels [181,183,184], albeit with differences regarding the size of the pore and the nature of the selectivity filter. Interestingly, a recent study suggests that the TRPV1 exhibits a phenomenon previously observed in P2X receptors [185,186] called pore dilation, in which the channel's selectivity for large cations is increased as compared to sodium ions in an agonist exposure-time-and concentration-dependent manner [187].…”

A Gate Hinge Controls the Epithelial Calcium Channel TRPV5

“…First, the molecular surface representation shows the localization and orientation of the D542 residue as a negatively charged ring at the entry of the channel (Fig. S2B)1126. Interestingly, the inner pore region contains the tryptophan residue (W583) residue at the intracellular end of TM6, with its side chain pointing towards the ion permeation pathway (Fig.…”

Adaptation of low-resolution methods for the study of yeast microsomal polytopic membrane proteins: a methodological review

“…Since its introduction by Koshland and co-workers [4], site-directed thiol chemistry has been exploited in many ways, such as to identify the residues that line the pathway taken by solutes across a membrane pore [5,37,38] and the amino acids forming the charge and solute specificity filters as well as the widths of these filters in such pores [5,39]; to monitor the binding of substrates to enzymes and transporters and the ensuing conformational changes; to study the topology of membrane proteins in different physiological states, for instance in different lipid environments [40]; to identify the residues involved in protein-protein interactions [41]; and to follow the transit of the passenger domain through the C-terminal β barrel of bacterial autotransporters [3]. Since its introduction by Koshland and co-workers [4], site-directed thiol chemistry has been exploited in many ways, such as to identify the residues that line the pathway taken by solutes across a membrane pore [5,37,38] and the amino acids forming the charge and solute specificity filters as well as the widths of these filters in such pores [5,39]; to monitor the binding of substrates to enzymes and transporters and the ensuing conformational changes; to study the topology of membrane proteins in different physiological states, for instance in different lipid environments [40]; to identify the residues involved in protein-protein interactions [41]; and to follow the transit of the passenger domain through the C-terminal β barrel of bacterial autotransporters [3].…”