Characterizing P2X2 Mutants Associated with Progressive Sensorineural Hearing Loss (DFNA41)

Abstract

P2X receptors are trimeric ion channels that open in response to extracellular ATP. Each subunit is comprised of intracellular N- and C-termini, a large extracellular domain containing the ATP binding site and two transmembrane (TM) helices that form a pore that is permeable to cations. P2X2 receptors are hypothesized to play a role in cochlear adaptation to elevated sound levels and protection from overstimulation. Whole-exome sequencing and linkage analysis of individuals with dominantly inherited progressive sensorineural hearing loss (DFNA41) revealed three different mutations of human P2X2 (hP2X2) receptor: V60L, D201Y, and G353R. To investigate the effects of mutations, recombinant hP2X2 receptors were expressed in HEK 293 cells and macroscopic currents were measured with the whole-cell patch clamp technique. We find that the G353R mutation lowers the apparent affinity for activation by ATP and produces a pronounced inward rectification, with large inward currents being observed only below −80 mV. In contrast, expression of the V60L mutation produces measurable constitutive currents with little rectification. To verify that the constitutive currents observed with V60L arise from the mutant receptor, we inserted a Cys into the pore-lining TM2 helix and observed robust and irreversible inhibition by thiol-reactive methanethiosulfonates (MTS). Interestingly, larger MTS reagents reacted more slowly with the V60L construct compared to control, suggesting that the mutation produces a structural alteration in the pore. Finally, the D201Y mutant channel does not respond to ATP and appears to be non-functional. Taken together, our results suggest that all three mutations greatly diminish or ablate ATP-activated currents at physiological voltages, and that the V60L also produces constitutively active channels. It will be interesting to further explore how these perturbations give rise to severe progression of hearing loss.