Outer Pore Topology of the ECaC-TRPV5 Channel by Cysteine Scan Mutagenesis

Abstract

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu 515 ) to the beginning of S6 (Ala 560 ). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro 527 and Ile 541 was compatible with the presence of a ␣-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions ؊1, 0, ؉1, ؉2 The TRP ion channels form a large class of cationic channels that are related to the product of the Drosophila TRP gene. TRP channels share a similar predicted topology of six transmembrane segments in which the amino acids that link the fifth and sixth transmembrane domains line the pore region (1). According to the recent IUPHAR classification of ion channels (2), the 21 members of the TRP family can be divided by sequence homology into three subfamilies (3, 4) as short (TRPCx), long or melastatin (TRPMx), and osm-9-like or vanilloid-like (TRPVx) channels. The molecular domains that are mostly conserved among TRP channels include part of the S6 segment, ankyrin repeats in the N terminus, and a "TRP domain" in the C terminus (EWKFAR) (5), the latter being absent from TRPV channels. The TRPC and TRPV proteins have 2-4 N-terminal ankyrin domains suggesting that these proteins are coupled to the spectrin-based membrane cytoskeleton.TRP channels vary significantly in their biophysical properties and gating mechanisms. In contrast to other members of the TRP family, TRPV5 and TRPV6 channels show strong inward rectification, exhibit anomalous mole-fraction effect, are activated by low [Ca 2ϩ ] i and inactivated by higher [Ca 2ϩ ] i (6 -8). TRPV5 and TRPV6 are also highly Ca 2ϩ -selective channels with PCa/PNa Ͼ 100. In particular, ECaC-TRPV5 displays a high Ca 2ϩ affinity with a K d of Ϸ2 M (7) that is comparable to the K d of Ϸ1 M for voltage-dependent Ca V channels (9). A single residue in the S5-S6 linker (Asp 542 ) was found to account for the high Ca 2ϩ affinity of ECaC-TRPV5 (7). The absence of the aspartate residue at the equivalent position in the pore region of TRPV1-4 channels might explain, together with the presence of a lysine residue, the Ϸ20-fold lower Ca 2ϩ selectivity of TRPV1-4 channels (10). TRPV5 and TRPV6 channels can also form homo-and heterotetramers suggesting that they are structurally and functionally related (11).There is currently very little structural data available on the pore architecture of Ca 2ϩ -selective TRP channels. It...

Uncoupling Proton Activation of Vanilloid Receptor TRPV1

“…Mutagenesis study in the pore region of TRPV1 produce functional changes in the permeation properties as expected (Garcia-Martinez et al, 2000). Cysteine accessibility experiments on a related homolog, TRPV5, support a coil structure followed by a helical segment and two distinct coiled fragments before S6 (Dodier et al, 2004). The present study also shows a close homology in the pore sequences after removal of 15 residues.…”

Active Ca2+ reabsorption in the connecting tubule

“…[51,75]. In addition, Huang and colleagues reported that PIP 2 activates TRPV5 and that activation of the channel by PIP 2 reduces the sensitivity of TRPV5 to the inhibition by the intracellular [Mg 2+ ] [17,42]. In this model, hydrolysis of PIP 2 by receptor activation of PLC may increase the sensitivity for Mg 2+ inhibition.…”

WNK3 positively regulates epithelial calcium channels TRPV5 and TRPV6 via a kinase-dependent pathway

“…Oocytes were coinjected with the S556C mutant of TRPV5, with or without WNK3. A previous study showed that TRPV5 proteins with cysteine at position 556 are inhibited when oocytes are incubated with the cell-impermeable thiol reagent 2-trimethylammonium-ethyl-methanethiosulfonate bromide (MTSET; Toronto Research Chemicals, North York, Ontario, Canada) (13). The oocytes were incubated with 2 mM MTSET for 5 min, which is sufficient to irreversibly inhibit the majority of functional TRPV5 channel proteins at the oocyte surface.…”