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

Abstract

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 pyridox-alphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), suramin, and 4,4′-diisothiocyanatostilbene-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 calciumchelating 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.

Mutation of the ATP-gated P2X 2 receptor leads to progressive hearing loss and increased susceptibility to noise

“…HEK293 cells were transfected with WT or mutant P2RX2 plasmid. Whole-cell recording was performed using an Axopatch 200B patch clamp amplifier (Molecular Devices) (15,16,18), and data were analyzed with jClamp.…”

“…P2X 2 receptors mediate a variety of cellular responses, including excitatory postsynaptic responses in sensory neurons (10,11). In the inner ear, P2X 2 receptors are thought to regulate sound transduction and auditory neurotransmission (12)(13)(14), outer hair cell electromotility (15,16), inner ear gap junctions (17), and K + recycling (17,18). We explored the effect of the inherited mutation in P2RX2 on function of the P2X 2 receptors and the consequences of loss of function of these ATP-gated channels on progressive and noise-induced hearing loss.…”

ATP-mediated potassium recycling in the cochlear supporting cells

“…1a) using an Axopatch 200B patch clamp amplifier (Molecular Devices, CA, USA). Patch pipettes were filled with an intracellular solution that contained (in mM) 140 KCl, 5 EGTA, 2 MgCl 2 , and 10 HEPES, pH 7.2, with initial resistance of [7][8][9]25]. The signal was filtered by a four-pole low-pass Bessel filter with a cutoff frequency of 2 kHz and digitized utilizing a Digidata 1322A (Molecular Devices, CA, USA).…”

“…A Y-tube perfusion system was used for the application of ATP and chemicals [25]. The potassium challenge was achieved by perfusion with high K + extracellular solutions, which were prepared by replacement of NaCl with KCl in the normal extracellular solution.…”

“…Both P2X and P2Y receptors are expressed in the cochlea, including supporting cells [16][17][18][19][20][21][22][23]. Recently, we have demonstrated that gap junctional hemichannels in the cochlear supporting cells can release ATP [24], which can activate P2x receptors in the outer hair cells (OHCs) to induce Ca ++ influx and modulate OHC electromotility [24,25]. P2X receptors have a cationic permeability, permeable to K + ions [15].…”