Differential Effects of Hearing Loss Mutations in Homomeric P2X2 and Heteromeric P2X2/3 Receptors

Abstract

P2X receptors are unspecific cation channels activated by ATP. They are expressed in various tissues and found in neuronal and immune cells. In mammals, seven subunits are described, which can assemble into homomeric and heteromeric trimers. P2X2 receptors play important roles in cochlear adaptation to elevated sound levels. Three mutations causing inherited progressive hearing loss have been identified. These mutations localize to the transmembrane domain 1 (V60L), the transmembrane domain 2 (G353R) and a β-sheet linking the ATP binding site to the pore (D273Y). Herein, mutations were studied in human homomeric P2X2 as well as in heteromeric P2X2/3 receptors. We measured their binding of a fluorescently labeled ATP derivative (fATP) and characterized the constructs using the patch-clamp technique. The conclusions from our results are as follows: 1. The mutations V60L and G353R show robust localization on the plasma membrane and binding of fATP, whereas the mutant D273Y has no binding to fATP. 2. The mutation V60L has an increased affinity to fATP compared with the wildtype. 3. The expression of hP2X2 V60L channels reduces cell viability, which may support its role in the pathogenesis of hearing loss. 4. All mutant P2X2 subunits can assemble into P2X2/3 heteromeric channels with distinct phenotypes.

Keywords: P2X receptors; fluorescent ATP; hearing loss mutations; heteromeric ion channels; ligand-binding assay; patch-clamp.

Figure 1

Structures of P2X receptors and hearing loss mutations. (A) Structure of a homomeric hP2X2 receptor and a heteromeric hP2X2/3 receptor containing one P2X2 and two P2X3 subunits in the background. One monomer with the three locations (red) of the mutations associated with sensorineural hearing loss is highlighted in front of the trimeric receptor. The structures were generated using alphafold3 and illustrated using chimera [7,11]. One ATP molecule (green) is shown in the binding pocket located between two subunits. The intracellular N- and C-termini are shortened for clarity. (B) Sequence alignment containing the 7 human P2X subunits and the P2X2 subunits from rats and mice. The sequences of the transmembrane domain 1 (TM1), a linker coupling the extracellular domain and the pore and transmembrane domain 2 (TM2) are shown. The sequences above are images of the secondary structures, illustrating a helix and a beta sheet (blue arrows). The locations of the mutations are highlighted in red. Conserved amino acids are in the grey background and labeled with an asterisk below the sequences.

Figure 2

Binding and activation of P2X wildtype receptors. (A–C) Representative confocal images of HEK 293 cells, stably expressing human P2X receptors, binding fATP at 1 µM (left). Quantification of the specific binding signal at 1 µM and 10 µM fATP. The bar graphs are derived from 15–40 images and represent the mean ± SEM (right). (D–F) Representative traces of recordings from stably expressing HEK 293 cells in the whole-cell configuration at different voltages in the presence of applicator-applied ATP (left) and ligand activation at a constant holding potential of −50 mV at the given ATP concentration (right). (G–I) Current–voltage relationships from −140 mV to +80 mV. One data point represents the mean ± SEM from 5–7 cells. ** p < 0.01; ns: not significant.

Figure 3

Binding and activation of P2X V60L mutant receptors. (A,B) Representative confocal images of HEK 293 cells, stably expressing human P2X receptors, binding fATP at 1 µM (left). Quantification of the specific binding signal at 1 µM and 10 µM fATP. Bar graphs are derived from 15–40 images and represent the mean ± SEM (right). (C,D) Representative traces of recordings from stably expressing HEK 293 cells in the whole-cell configuration at different voltages in the presence of applicator-applied ATP (left) and ligand activation at a constant holding potential of −50 mV at the given ATP concentration (right). (E,F) Current–voltage relationships from −140 mV to +80 mV. One data point represents the mean ± SEM from 5–9 cells. (G) Deactivation time constants derived from exponential fits after removal of 100 µM ATP are shown as the mean ± SEM. (H) Viability of the cells was expressed as the normalized luminescence signal from the luciferase reaction after 48 h of tetracycline treatment; 12 wells in a 96-well plate format were analyzed. T: tetracycline treatment. ** p < 0.01; **** p < 0.0001; ns: not significant.

Figure 4

Binding and gating of P2X D273Y mutant receptors. (A,B) Representative confocal images of HEK 293 cells, stably expressing human P2X receptors, binding fATP at 1 µM. (C) Quantification of the specific binding signal at 1 µM and 10 µM fATP. The bar graphs are derived from 15–40 images and represent the mean ± SEM (right). (D,E) Representative traces of recordings from stably expressing HEK cells in the whole-cell configuration at different voltages in the presence of applicator-applied ATP (left) and ligand activation at a constant holding potential of −50 mV at the given ATP concentration (right). (F,G) Current–voltage relationships from −140 mV to +80 mV. One data point represents the mean ± SEM from 5–13 cells. * p < 0.05; ns: not significant.

Figure 5

Binding and activation of P2X G353R mutant receptors. (A,B) Representative confocal images of HEK 293 cells, stably expressing human P2X receptors, binding fATP at 1 µM (left). Quantification of the specific binding signal at 1 µM and 10 µM fATP. The bar graphs are derived from 15–40 images and represent the mean ± SEM (right). (C–E) Representative traces of recordings from stably expressing HEK 293 cells in the whole-cell configuration at different voltages in the presence of applicator-applied ATP (left) and ligand activation at a constant holding potential of −50 mV at the given ATP concentration (right). (F–H) Normalized current–voltage relationships from −140 mV to +80 mV. One data point represents the mean ± SEM from 5–9 cells. The maximum amplitude of the current signals was normalized with respect to the maximum current amplitude of 100 µM ATP at −140 mV. *** p < 0.001; **** p < 0.0001.