Background
While voltage-gated Na+ and Ca2+ human channelopathies have been extensively studied, the role of transient receptor potential (TRP) channels in human Mendelian pain disorders is less established. TRP channels are implicated in several aspects of sensation [1] and knockout animal models suggest that TRP channels also mediates pain perception and sensitivity [2]. The only human TRPA1-channelopathy so far described is associated with the gain-of-function N855S mutation, causing an autosomal dominant familial episodic pain syndrome [3]. We identified a novel TRPA1 variant (A172V) in a patient/study participant with an episodic somatic pain disorder. The whole study involved a cohort of 220 patients with a clinical history of neuropathic pain disorders, who underwent whole genome sequencing as part of the NIHR-Bioresource. In silico analysis have predicted that the A172V variant is likely to have a significant impact on the protein function, highlighting it as a promising candidate for functional studies.
Methods
In silico prediction analysis were employed to evaluate the variant position, the minor allele frequency and the concordant pathogenicity, via multiple in silico algorithms such as SIFT, polyphen and Align GDVD. In vitro functional studies involved the electrophysiological characterisation of the hTRPA1 A172V variant.
Results
The novel A172V mutation is located in the ankyrin repeat domain region of TRPA1, which appears important for the channel activation [4]. Our electrophysiological data have shown that heterologous over-expression of the A172V variant results in a gain of function, in response to TRPA1 agonists. In the presence of 25 µM AITC, A172V showed a pronounced linearisation of the current-voltage relationship, with a steeper activation curve observed at positive potentials compared to WT (+100mV, WT = 21.12 ± 2.46pA/pF n=13, A172V= 56.92 ± 12.52pA/pF n=9, p<0.005). Currents were significantly increased at positive potentials, and analysis of tail currents demonstrated a significant leftward shift of voltage dependence of channel activation (V1/2 = 35.55 ± 5.45mV, and 59.16 ± 5.25mV, p<0.05, for A172V and WT, respectively). We also compared the biophysical properties of A172V to those of N855S in response to AITC. After agonist application, N855S revealed increased outward and inward currents, and overall a more pronounced voltage-sensitivity than A172V (+100mV, N855S = 38.12 ± 10.77pA/pF; -100mV, N855S = -13.48 ± 2.40pA/pF, V1/2 = 37.03 ± 8.28mV, n=12). Similar conclusions were observed after application of 100 µM Menthol, a non-electrophilic TRPA1 agonist (V1/2, WT = 61.83 ± 7.37mV n = 8, A172V = 36.41 ± 7.58mV n = 9, N855S = 33.60 ± 6.67mV n = 6, p<0.05). These data confirm the strong phenotype previously reported for N855S, and reveal an intermediate phenotype for the A172V variant.
Conclusions
Whole genome sequencing has revealed a further human TRPA1 (A172V) pain channelopathy. This was associated with episodic truncal pain and the A172V variant resulted in gain of function of TRPA1, demonstrating the role of ankyrin repeat domain in mediating the gating properties of TRPA1. In future studies, we will be examining the role of TRPA1 variants in determining the risk and severity of neuropathic pain due to sensory neuropathy.