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.
Centre for Cardiovascular and Metabolic Research, Hull-York Medical School, University of Hull, Daisy Building, Castle Hill Hospital, Cottingham, Hull HU16 5JQ, U.K. k.morgan@hull.ac.uk.
Kevin
K
Morgan
Centre for Cardiovascular and Metabolic Research, Hull-York Medical School, University of Hull, Daisy Building, Castle Hill Hospital, Cottingham, Hull HU16 5JQ, U.K.
Laura Rachel
LR
Sadofsky
Centre for Cardiovascular and Metabolic Research, Hull-York Medical School, University of Hull, Daisy Building, Castle Hill Hospital, Cottingham, Hull HU16 5JQ, U.K.
Alyn Hugh
AH
Morice
D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics*; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017434:Protein Structure, Tertiary; D000074025:TRPA1 Cation Channel; D050053:TRPM Cation Channels / Q000737:chemistry / Q000235:genetics*; D050051:Transient Receptor Potential Channels / Q000737:chemistry / Q000235:genetics*
D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050053:TRPM Cation Channels; C425966:TRPM8 protein, human; D050051:Transient Receptor Potential Channels
expression levels; human TRP channels; single nucleotide polymorphisms (SNPs)