The Single Pore Residue Asp542 Determines Ca2+ Permeation and Mg2+ Block of the Epithelial Ca2+ Channel

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, ...

Section: Discussionmentioning

confidence: 99%

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

Kühn

Knop²,

Lückhoff

2007

“…However, the only negatively charged amino acid residues in the putative pore-forming region of TRPV6 are a glutamate residue at position 535 and two aspartate residues at positions 542 and 550. All three amino acids are conserved in the TRPV5 protein from rabbit and especially the aspartate residue at position 542 has been shown to decisively affect Ca 2ϩ permeation (27,28). Substituting aspartate at position 542 versus an alanine in human TRPV6 abolished channel activity (10), which was also the case if this pore mutant was C-terminally EGFP-tagged.…”

Section: Discussionmentioning

confidence: 99%

Voltage-dependent Changes of TRPV6-mediated Ca2+ Currents

Bödding

2005

“…All of the nonpolar amino acids were replaced by a polar amino acid, Ser, and the polar amino acids were replaced by a small, nonpolar 1 The abbreviations used are: GFP, green fluorescence protein; GST, glutathione S-transferase. amino acid, Ala. Gly is classified as a polar amino acid and was thus replaced by Ala. Because it has been reported that the negatively charged amino acids Glu and Asp in the transmembrane domain contribute to ion selectivity in voltage-gated Ca 2ϩ channels (30,31) and ECaC (32,33) and to interaction with channel blockers (34,35), the remarkable Asp 341 was replaced by each of 7 amino acids, Glu, Lys, Arg, Thr, Asn, Ala, and Leu, and the positively charged amino acid His 353 was also replaced by each of 3 amino acids, Asp, Arg, and Ala. The mutant genes bearing these single amino acid substitutions were then introduced into cells of the mid1-⌬5 mutant, and the resulting transformants were examined for viability and Ca 2ϩ accumulation after exposure to ␣-factor.…”

Section: H3 Is Essential For Mid1mentioning

confidence: 99%

Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

Tada¹,

Ohmori²,

Iida³

2003

The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca 2؉ -permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the ␣1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro sitedirected mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca 2؉ accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca 2؉ accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.