ATP activates P2x receptors and requires extracellular Ca⁺⁺ participation to modify outer hair cell nonlinear capacitance

Intracochlear ATP is an important mediator in regulating hearing function. ATP can activate ionotropic purinergic (P2x) and metabotropic purinergic (P2y) receptors to influence cell functions. In this paper, we report that ATP can activate P2x receptors directly to modify outer hair cell (OHC) electromotility, which is an active cochlear amplifier determining hearing sensitivity and frequency selectivity in mammals. We found that ATP, but not UTP, a P2y receptor agonist, reduced the OHC electromotility-associated nonlinear capacitance (NLC) and shifted its voltage dependence to the right (depolarizing) direction. Blockage of the activation of P2x receptors by pyridoxalphosphate-6-azophenyl- 2′,4′-disulfonic acid (PPADS), suramin, and 4,4′- diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS) could block the ATP effect. This modification also required extracellular Ca⁺⁺ participation. Removal of extracellular Ca⁺⁺ abolished the ATP effect. However, chelation of intracellular Ca⁺⁺ concentration by a fast calcium-chelating reagent 1,2-bis(o-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid (BAPTA, 10 mM) did not affect the effect of ATP on NLC. The effect is also independent of K⁺ ions. Substitution of Cs⁺ for intracellular or extracellular K⁺ did not affect the ATP effect. Our findings indicate that ATP activates P2x receptors instead of P2y receptors to modify OHC electromotility. Extracellular Ca⁺⁺ is required for this modification.