Genetic variants affecting human TRPA1 or TRPM8 structure can be classified in vitro as 'well expressed', 'poorly expressed' or 'salvageable'.

Multiple mis-sense variants of TRPA1 (transient receptor potential A1) and TRPM8 (transient receptor potential M8) are recorded in the human genome single nt polymorphism (SNP) database, but their potential impact on channel signalling in patho-physiology is not fully explored. Variants, mostly quite rare in the general human population, alter sites in different structural domains of these homo-tetrameric ion channel proteins. The effects of individual SNPs affecting the large cytoplasmic N-terminal domain have not been completely documented for TRPM8 or TRPA1. We examined the Ca(2+) signalling properties of a short-list of eight variants affecting the N-terminal domain by individual expression in human embryonic kidney HEK293 or neuroblastoma (SH-SY5Y) cell lines (four SNP variants for TRPM8: G150R, K423N, R475C, R485W and four for TRPA1: Y69C, A366D, E477K, D573A). These were compared with TRPA1 SNP variants affecting intracellular loops located beyond the N-terminal domain and associated with gain of function (such as increased sensitivity to agonists: TRPA1 R797T and N855S). A substitution in TRPA1 (Y69C) exhibited high expression/sensitivity to agonists (high iCa(2+)max (maximum level of intracellular calcium ion), similar to R797T, but less sensitive than N855S), whereas each of the other non-conservative substitutions exhibited poor signalling response (low iCa(2+)max). Responses from these poorly expressed variants could be salvaged, to different extents, by pre-treating cells with the Src (Src protein) family inhibitor protein kinase inhibitor PP2 (PP2: 4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), or with micromolar Zn(2+). The TRPA1 variants and several experimental mutants (TRPA1 Y97F, Y226F and YY654-655FF) expressed poorly in SH-SY5Y compared with HEK293 cells. More in-depth studies are needed to identify SNP variants eliciting gain of function in these TRP (transient receptor potential) channels and to assess their roles in medical conditions.

Morgan K ，Sadofsky LR ，Morice AH 《-》

Human TRPM8 and TRPA1 pain channels, including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor.

Morgan K ，Sadofsky LR ，Crow C ，Morice AH ... -  《-》

Structural modeling and patch-clamp analysis of pain-related mutation TRPA1-N855S reveal inter-subunit salt bridges stabilizing the channel open state.

The ankyrin transient receptor potential channel TRPA1 is a polymodal sensor for noxious stimuli, and hence a promising target for treating chronic pain. This tetrameric six-transmembrane segment (S1-S6) channel can be activated by various pungent chemicals, such as allyl isothiocyanate or cinnamaldehyde, but also by intracellular Ca(2+) or depolarizing voltages. Within the S4-S5 linker of human TRPA1, a gain-of-function mutation, N855S, was recently found to underlie familial episodic pain syndrome, manifested by bouts of severe upper body pain, triggered by physical stress, fasting, or cold. To clarify the structural basis for this channelopathy, we derive a structural model of TRPA1 by combining homology modeling, molecular dynamics simulations, point mutagenesis and electrophysiology. In the vicinity of N855, the model reveals inter-subunit salt bridges between E854 and K868. Using the heterologous expression of recombinant wild-type and mutant TRPA1 channels in HEK293T cells, we indeed found that the charge-reversal mutants E854R and K868E exhibited dramatically reduced responses to chemical and voltage stimuli, whereas the charge-swapping mutation E854R/K868E substantially rescued their functionalities. Moreover, mutation analysis of highly conserved charged residues within the S4-S5 region revealed a gain-of-function phenotype for R852E with an increased basal channel activity, a loss of Ca(2+)-induced potentiation and an accelerated Ca(2+)-dependent inactivation. Based on the model and on a comparison with the recently revealed atomic-level structure of the related channel TRPV1, we propose that inter-subunit salt bridges between adjacent S4-S5 regions are crucial for stabilizing the conformations associated with chemically and voltage-induced gating of the TRPA1 ion channel.

Zíma V ，Witschas K ，Hynkova A ，Zímová L ，Barvík I ，Vlachova V ... -  《-》

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells.

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression.

Louhivuori LM ，Bart G ，Larsson KP ，Louhivuori V ，Näsman J ，Nordström T ，Koivisto AP ，Akerman KE ... -  《-》

Overexpression of human transient receptor potential M5 upregulates endogenous human transient receptor potential A1 in a stable HEK cell line.

Buber MT ，Cerne R ，Cortés RY ，Bryant RW ，Lee SP ... -  《-》