Dataset Information

Importance of a conserved sequence motif in transmembrane segment S3 for the gating of human TRPM8 and TRPM2.

ABSTRACT: For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles of the motif for the voltage-dependent and voltage-independent gating, we performed reciprocal replacements of the asparagine and aspartate within the S3 motif in both channels, following the proposed idea that specific electrostatic interactions with other domains take place during gating. Wild-type and mutant channels were heterologeously expressed in HEK-293 cells and channel function was analyzed by whole-cell patch-clamp analysis as well as by Ca(2+)-imaging. Additionally, the expression of the channels in the plasma membrane was tested by Western blot analysis, in part after biotinylation. For the mutations of TRPM8, responses to menthol were only compromised if also the expression of the glycosylated channel isoform was prevented. In contrast, responses to cold were consistently and significantly attenuated but not completely abolished. For TRPM2, surface expression was not significantly affected by any of the mutations but channel function was only retained in one variant. Remarkably, this was the variant of which the corresponding mutation in TRPM8 exerted the most negative effects both on channel function and expression. Furthermore, we performed an exchange of the inner pair of residues of the N-x-x-D motif between the two channels, which proved deleterious for the functional expression of TRPM8 but ineffective on TRPM2. In conclusion, the N-x-x-D motif plays specific roles in TRPM8 and TRPM2, reflecting different requirements for voltage-dependent and voltage-independent channel gating.

SUBMITTER: Winking M

PROVIDER: S-EPMC3503820 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Importance of a conserved sequence motif in transmembrane segment S3 for the gating of human TRPM8 and TRPM2.

Winking Mathis M Hoffmann Daniel C DC Kühn Cornelia C Hilgers Ralf-Dieter RD Lückhoff Andreas A Kühn Frank J P FJ

PloS one 20121121 11

For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles ...[more]

PMID: 23185472

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Project description:Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.

Project description:Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.

Dataset Information

Importance of a conserved sequence motif in transmembrane segment S3 for the gating of human TRPM8 and TRPM2.

Publications

Importance of a conserved sequence motif in transmembrane segment S3 for the gating of human TRPM8 and TRPM2.

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