Missense variant analysis in the TRPV1 ARD reveals the unexpected functional significance of a methionine

The Transient Receptor Potential Vanilloid sub-type 1 (TRPV1) is an ion channel implicated in various aspects of inflammatory pain. Whereas ion conduction and the binding of vanilloids and many other important ligands occur within the transmembrane domain, the role of the large cytoplasmic N-terminal ankyrin repeat domain in channel function is unclear. We have previously described that the number of distinct amino acid variants observed in the human population at each TRPV1 N-terminal ankyrin repeat domain position is positively correlated with their solvent accessible surface area (SASA). Our prior study led us to hypothesize that sites with strong deviations from the correlation are likely to have functional relevance. In this study, we isolated two buried residues (M308/A256) in the ARD that have low SASA scores and yet at least 3 or 4 alternate types of amino acids are found within the human population at these two positions, setting them apart from most other sites with similarly low SASA for which no human missense variants have been found. Side chains from both residues extend into a buried pocket and are within 5 Å of each other in a closed channel structure configuration. Moreover, despite its variability among the human population, M308 is highly conserved across species as well as across TRPV subfamily members. To address these apparently contradictory observations, we used mutagenesis to examine the functional role of M308. We determined using electrophysiology that substitutions at position M308 which preserve or reduce side-chain volume had no effect on channel function, whereas substitutions with the larger residues resulted in either complete loss of channel activity or increased activity in response to both capsaicin and temperature. We confirmed that M308 is a partially buried site since M308C did not show any evidence of reactivity to various cysteine-modifying reagents. We observed that converting the methionine to histidine bestows a pH-dependent effect on the channel that is different from wild type, consistent with the side-chain at position 308 exerting a functional effect on the channel either sterically or electrostatically by action of protonation. These observations establish that steric or electrostatic perturbations at position M308 influence channel activity, but that a methionine is not required for normal channel function, raising the question of why M308 is so highly conserved. We speculate that given the functional effect of perturbations introduced at position 308, M308 could have a conserved role in redox signaling as a target for oxidation-dependent side-chain covalent modification. On the other hand, M308 could be used as an alternative ‘start’ codon, which is consistent with a short 5’ splice variant of TRPV1 that has been reported in the literature. We show that the truncated splice variant significantly diminishes surface expression of full-length TRPV1 when co-expressed in HEK293 cells. Together, our findings reveal a functionally important conserved site within the ARD of TRPV1 that could have physiologically relevant roles in oxidation-dependent channel regulation as well as tuning the number of active channels in the membrane by enabling expression of a shorter dominant-negative splice variant.