ABSTRACT
Protein structures and mutagenesis studies have been instrumental in elucidating molecular mechanisms of ion channel function, but making informed choices about which residues to target for mutagenesis can be challenging. Therefore, we investigated the potential for using human population genomic data to further refine our selection of mutagenesis sites in TRPV1. Single nucleotide polymorphism data of TRPV1 from gnomAD 2.1.1 revealed a lower number of missense variants within buried residues of the ankyrin repeat domain and an increased number of variants between secondary structure elements of the transmembrane segments. We hypothesized that residues critical to interactions at interfaces between subunits or domains in the channel would exhibit a similar reduction in variants. We identified in the structure of ground squirrel TRPV1 (PDB: 7LQY) a possible electrostatic network between K155 and K160 in the N-terminal ankyrin repeat domain and E761 and D762 in the C-terminus (K-KED). Consistent with our hypothesis for residues at key interface sites, none of the four residues have any variants reported in gnomAD 2.1.1. Ca2+ imaging of TRPV1 K-KED mutants confirmed significant roles for these residues, but we found that the electrostatic interaction is not essential since channel function is still observed in total charge reversals on the C-terminal side of the interface (E761K/D762K). Interestingly, Ca2+ imaging responses for a charge swap experiment with K155D/D762K showed partially restored wild-type responses. Using electrophysiology, we found that charge reversals on either K155 or D762 increased the baseline currents of TRPV1, and the charge swapped double mutant, K155D/D762K, partially restored baseline currents to wild-type levels. We interpret these results to mean that contacts across residues in the K-KED interface shift the equilibria of conformations to closed pore states. Our study demonstrates the utility and applicability of a combined missense variant and structure targeted investigation of residues at TRPV1 subunit interfaces.
