The Transmembrane Segment S6 Determines Cation versus Anion Selectivity of TRPM2 and TRPM8

Abstract

TRPM2 and TRPM8, closely related members of the transient receptor potential (TRP) family, are cation channels activated by quite different mechanisms. Their transmembrane segments S5 and S6 are highly conserved. To identify common structures in S5 and S6 that govern interaction with the pore, we created a chimera in which the S5-pore-S6 region of TRPM8 was inserted into TRPM2, along with a lysine at each transition site. Currents through this chimera were induced by ADP-ribose (ADPR) in cooperation with Ca 2؉ . In contrast to wild-type TRPM2 channels, currents through the chimera were carried by Cl ؊ , as demonstrated in ion substitution experiments using the cation N-methyl-D-glucamine (NMDG) and the anion glutamate. Extracellular NMDG had no effects. The substitution of either intracellular or extracellular Cl ؊ with glutamate shifted the reversal potential, decreased the current amplitude and induced a voltage-dependent block relieved by depolarization. The lysine in S6 was responsible for the anion selectivity; insertion of a lysine into corresponding sites within S6 of either TRPM2 or TRPM8 created anion channels that were activated by ADPR (TRPM2 I1045K) or by cold temperatures (TRPM8 V976K). The positive charge of the lysine was decisive for the glutamate block because the mutant TRPM2 I1045H displayed cation currents that were blocked at acidic but not alkaline intracellular pH values. We conclude that the distal part of S6 is crucial for the discrimination of charge. Because of the high homology of S6 in the whole TRP family, this new role of S6 may apply to further TRP channels. The members of the transient receptor potential (TRP)3 family of non-selective cation channels display an extraordinarily broad spectrum of activation mechanisms reflecting their involvement in manifold biological processes. The basic architecture of TRP channels is shared with the well characterized voltage-gated K ϩ channels. Here, the transmembrane segment S6 contains the activation gate that opens the pore in response to the movement of the activated S4 voltage sensor (1, 2). Although TRP channels do not contain a classical voltage sensor, one may presume an interaction of S6 with the pore that governs functional relevance for the properties of TRP channels.TRPM2 and TRPM8 of the melastatin-related subfamily of TRP channels are the closest relatives within the TRP family (42% identical residues, Ref.3) but their activation mechanisms are completely different. The principal activators of TRPM2 are intracellular ADP-ribose (ADPR) and reactive oxygen species such as hydrogen peroxide (4, 5). Accordingly, TRPM2 is involved in the cellular responses to oxidative and nitrative stress (6 -14).The TRPM8 channel mediates the cold sensation of the somatosensory system (3, 15, 16) but was initially discovered as a protein with up-regulated expression in prostate cancer and some other malignant tissues; these roles of TRPM8 are apparently independent of any significant temperature variations (17). TRPM8 is gated by cold temperatures, ...

Contribution of the S5-Pore-S6 Domain to the Gating Characteristics of the Cation Channels TRPM2 and TRPM8

“…It should be noted that this chimera differs from a previously characterized one that was anion-selective due to the single amino acid exchange I1045K (indicated in Fig. 1) (19). In whole-cell experiments with ADPR (0.05-0.6 mM) added to the pipette fluid or after extracellular stimulation with 10 mM hydrogen peroxide, this chimera displayed cation-selective currents with a linear I-V relation that were readily blocked in the inward direction by NMDG.…”

“…The corresponding amino acid sequences of TRPM2 and TRPM8 are shown in single-letter code, and conserved residues are shown in boldface. The site within S6 where the exchange of a hydrophobic residue with a lysine reverses the charge selectivity of the pore in both TRPM2 and TRPM8 is marked with an asterisk (19). mean open time was 293 Ϯ 11 ms (Fig.…”

The A563T variation of the renal epithelial calcium channel TRPV5 among African Americans enhances calcium influx

“…Because the 563 position in the last transmembrane domain of TRPV5 is close to Asp 542 , it is likely that A563T substitution may affect the architecture of the pore and make it more accommodating for Ca 2ϩ ion permeation, although the lack of an accurate three-dimensional structure of TRPV5 precludes a precise prediction how this happens. It has been recently demonstrated that the substitution of a hydrophobic amino acid residue with a positively charged lysine in the last transmembrane domain of TRPM2 and TRPM8 converts the channels from cation selective to anion selective (20). This suggests that amino acid residues in the last transmembrane domain may participate in defining ion permeation properties directly or indirectly by affecting the conformation of the amino acid residues in the cation filter at the adjacent pore region.…”

Pharmacology of transient receptor potential melastatin channels in the vasculature

“…TM4 and the TM4-TM5 linker in TRPM8 determines its sensitivity to voltage, temperature and menthol , while the distal part of TM6 determines cation versus anion selectivity, at least in TRPM2 and TRPM8 channels (Kuhn et al, 2007).…”