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 Ca2+-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.

Positive selection acted on the extracellular transmembrane linkers of heat receptors during evolution

“…It is understandable that no selection occurred in the transmembrane segments because any change in these would destroy the transmembrane structure (Winking et al, 2012). Interestingly, no selected site was identified in the intracellular linkers between S2 and S3, and between S4 and S5, further supporting our notion that the external side is important for responding to temperature alterations.…”

“…It has also been suggested that the extracellular Ploop is a critical structure for temperature sensitivity (Winking et al, 2012;Yang et al, 2010). In present study, we investigated the molecular evolution of thermo-TRP channel genes across vertebrate phylogeny and addressed the question of how natural selection has acted on the structure and functions of cold and heat receptors in order to understand how animal preferences for hot versus cold sensitivity have evolved.…”

Voltage vs. Ligand I: Structural basis of the intrinsic flexibility of S3 segment and its significance in ion channel activation

“…The NxxD motif could represent a sensor in mechanosensation since mutant channels shift the activation curves to membrane tensions less than those required to activate the wild-type MscL [43]. As this motif is also a functional component of the V-sensor in voltagedependent channels, mutations of this region are likewise critical in channel gating [27,44,45]. Interestingly, in MscL orthologues, two helical turns below the NxxD motif, a conserved Gly residue (G24 in M. tuberculosis) forms a girdle surrounding the narrowest portion of the channel lumen [46].…”

“…In TRP channels, which are weakly voltagesensitive but strongly activated by stimuli as diverse as heat, cold, and several ligands, gating is also achieved through conformational changes facilitated by local flexibility. These motions are associated with specific regions of the VSD, including the S3 segment, as well as linkers S3-S4L and S4-S5L [9,[27][28][29]. In CNG channels, which are virtually voltage-independent, the opening of the pore has evolved in strict dependence on the cyclic nucleotide-binding through conformational changes involving the S4-S5L [30].…”

Sperm specific expression of temperature-sensitive ion channel TRPM8 correlates with vertebrate evolution

“…TRPM8 also act as a voltage gated ion channel which becomes activated upon membrane depolarization [32][33][34]. Indeed, a small yet highly conserved (in many ion channels) region located in the transmembrane region 3 (TM3) of TRPM8 can act as a "voltage-sensor" [35]. Similarly, a small double cysteine motif is important for TRPM8 channel functions [36].…”