Nicholas P. Raybould

Purinergic Regulation of Sound Transduction and Auditory Neurotransmission

In the cochlea, extracellular ATP influences the endocochlear potential, micromechanics, and neurotransmission via P2 receptors. Evidence for this arises from studies demonstrating widespread expression of ATP-gated ion channels (assembled from P2X receptor subunits) and G protein-coupled receptors (P2Y receptors). P2X2 receptor subunits are localized to the luminal membranes of epithelial cells and hair cells lining scala media. These ion channels provide a shunt pathway for K+ ion egress. Thus, when noise exposure elevates ATP levels in this cochlear compartment, the K+ conductance through P2X receptors reduces the endocochlear potential. ATP-mediated K+ efflux from scala media is complemented by a P2Y receptor G protein-coupled pathway that provides coincident reduction of K+ transport into scala media from the stria vascularis when autocrine or paracrine ATP signalling is invoked. This purinergic signalling likely provides a basis for a reactive homoeostatic regulatory mechanism limiting cochlear sensitivity under stressor conditions. Elevation of ATP in the perilymphatic compartment under such conditions is also likely to invoke purinergic receptor-mediated changes in supporting cell micromechanics, mediated by Ca2+ influx and gating of Ca2+ stores. Independent of these humoral actions, ATP can be classified as a putative auditory neurotransmitter based on the localization of P2X receptors at the spiral ganglion neuron-hair cell synapse, and functional verification of ATP-gated currents in spiral ganglion neurons in situ. Expression of P2X receptors by type II spiral ganglion neurons supports a role for ATP as a transmitter encoding the dynamic state of the cochlear amplifier.

Variation in expression of the outer hair cell P2X receptor conductance along the guinea‐pig cochlea.

1. Whole‐cell patch‐clamp recordings were used to determine the variation in the P2X receptor conductance, activated by extracellular ATP, in outer hair cells (OHCs) isolated from each of the four turns of the guinea‐pig cochlea. 2. In standard solution (containing 1.5 mM Ca2+) slope conductances were determined in OHCs of known origin from current‐voltage relationships obtained from voltage ramps applied between ‐100 and +50 mV. Membrane conductance throughout this voltage range was greatest in OHCs originating from the basal (high frequency encoding) region of the cochlea. This gradient in OHC conductance from apex to base of the cochlea can be attributed to variation in expression of both a negatively activated K+ conductance and a TEA‐sensitive outwardly rectifying K+ conductance. OHC slope conductance measured about a membrane potential of ‐75 mV increased from a mean of 33.5 nS in the apical region (turn 4) to 96.8 nS in the basal region (turn 1) of the cochlea. 3. Removal of external Ca2+ reduced OHC conductance by an average of 25%, reflecting a Ca2+ dependence of the background conductances in these cells. In zero external Ca2+ the mean slope conductance measured at ‐75 mV in the apical turn was 25.0 nS compared with 73.8 nS in the basal turn. 4. In Ca(2+)‐free solution both 2 mM and 4 microM ATP produced inward currents that were progressively larger in OHCs originating from more basal regions of the cochlea. The steady‐state inward current elicited by 2 mM extracellular ATP increased from ‐1.44 to ‐3.26 nA for turns 4 and 1, respectively. 5. The P2X receptor conductance was determined between ‐100 and +50 mV by comparing voltage ramps in the presence and absence of extracellular ATP in Ca(2+)‐free solution. The conductance was inwardly rectifying with a reversal potential close to 0 mV. Measured close to the resting membrane potential of the cells (‐75 mV), 2 mM ATP elicited an average 300% increase in conductance in parallel with the systematic increase in background conductance which occurs in OHCs originating from the more basal regions of the cochlea. The conductance at ‐75 mV activated by 2 mM ATP increased from a mean of 59.6 nS in turn 4 OHCs to a mean of 166.2 nS in turn 1 OHCs. The conductance activated by 4 microM ATP was also greater in the basal turn OHCs (45.3 nS) than in the apical region OHCs (5.9 nS). 6. The number of ATP‐gated ion channels on individual OHCs, presumed to be localized to the stereocilia, increases from approximately 6000 in turn 4 cells to 16,500 in turn 1 cells, based on estimates of unitary conductance and average maximum ATP‐activated OHC conductance (2 mM ATP).

ATP-gated ion channel expression in primary auditory neurones

Extracellular ATP acts via ionotropic P2X receptors to mediate fast neurotransmission in the central and autonomic nervous systems. Recent data, including identification of P2X2 receptor mRNA expression by spiral ganglion neurones, suggests that purinergic signalling may influence auditory neurotransmission via ATP-gated ion channels assembled from these subunits. Expression of the P2X2 receptor was localized to the region of the spiral ganglion neurone synapses with the inner hair cells using a P2X2 receptor specific antiserum. Whole-cell patch clamping of neurones cultured from post-natal day 3-5 spiral ganglia demonstrated a heterogeneity of ATP-activated conductances, consistent with the functional expression of P2X2 receptor subunit isoforms along with possible co-expression of additional P2X receptor subunits. These data provide substantive support for a purinergic transmission element at the peripheral auditory synapse.

TRPC-like conductance mediates restoration of intracellular Ca2+ in cochlear outer hair cells in the guinea pig and rat.

Ca2+ signalling is central to cochlear sensory hair cell physiology through its influence on sound transduction, membrane filter properties and neurotransmission. However, the mechanism for establishing Ca2+ homeostasis in these cells remains unresolved. Canonical transient receptor potential (TRPC) Ca2+ entry channels provide an important pathway for maintaining intracellular Ca2+ levels. TRPC3 subunit expression was detected in guinea pig and rat organ of Corti by RT‐PCR, and localized to the sensory and neural poles of the inner and outer hair cells (OHCs) by confocal immunofluorescence imaging. A cation entry current with a TRPC‐like phenotype was identified in guinea pig and rat OHCs by whole‐cell voltage clamp. This slowly activating current was induced by the lowering of cytosolic Ca2+ levels ([Ca2+]i) following a period in nominally Ca2+‐free solution. Activation was dependent upon the [Ca2+]o and was sustained until [Ca2+]i was restored. Ca2+ entry was confirmed by confocal fluorescence imaging, and rapidly recruited secondary charybdotoxin‐ and apamin‐sensitive KCa currents. Dual activation by the G protein‐coupled receptor (GPCR)–phospholipase C–diacylglycerol (DAG) second messenger pathway was confirmed using the analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG). Ion substitution experiments showed that the putative TRPC Ca2+ entry current was selective for Na+ > K+ with a ratio of 1: 0.6. The Ca2+ entry current was inhibited by the TRPC channel blocker 2‐aminoethyl diphenylborate (2APB) and the tyrosine kinase inhibitor, erbstatin analogue. We conclude that TRPC Ca2+ entry channels, most likely incorporating TRPC3 subunits, support cochlear hair cell Ca2+ homeostasis and GPCR signalling.

Positional Analysis of Guinea Pig Inner Hair Cell Membrane Conductances: Implications for Regulation of the Membrane Filter

In mammals, sound transduction by inner hair cells (IHC) generates a receptor potential whose amplitude and phase drive auditory nerve firing. The membrane filter properties that define the input-output function of IHC are derived from membrane conductance and capacitance. These elements of the membrane filter were quantified using whole-cell voltage clamp of IHC from the four turns of the guinea pig cochlea. IHC membrane properties were remarkably constant along the cochlea, in contrast with all other auditory hair cell systems, and suggests that extrinsic processes such as the active filter provided by the outer hair cells are matched to a constant transfer function of the IHC. Two outwardly rectifying K+ currents contribute to the IHC membrane conductance. These combined currents activate at approximately -55 mV. IHC mean input resistance was 140 MW and capacitance was 10.0 pF, generating a membrane time constant of 1.4 ms or a corner frequency of approximately 115 Hz, which is consistent with reported low-frequency roll-off of the IHC AC receptor potential in vivo. Approximately 40% of the 313 nS total K+ conductance about 0 mV was attributed to charybdotoxin-sensitive KCa channels (also sensitive to cell dialysis with the Ca2+ chelator BAPTA or removal of extracellular Ca2+). The only known ligand-activated conductance in mature IHC, the P2X receptor conductance, averaged 31 nS (activated by 400 mM ATP; about -75 mV) irrespective of cell origin. Thus, regulation of intracellular Ca2+ and activation of P2X receptors by extracellular ATP provide capacity for local dynamic fine-tuning of the IHC membrane filter.

Allosteric modulation of native cochlear P2X receptors: insights from comparison with recombinant P2X2 receptors.

Extracellular adenosine 5′-triphosphate (ATP)-gated ion channels assembled from P2X receptor subunits exhibit subunit-selective allosteric modulation by protons and divalent cations. In voltage-clamped guinea-pig cochlear outer hair cells (OHC) and Deiters’ cells (DC), H+ and Cu2+, but not Zn2+, enhanced the P2X receptor-mediated inward currents. Acid pH (6.5) potentiated OHC ATP-gated currents by 45%. Co-application of Cu2+ (1–40 µM) with ATP increased the response by 20%. In DCs, ATP-gated currents were potentiated 85% by acid pH, and 70% by Cu2+. Alkaline pH inhibited ATP-gated inward currents by 73% in OHCs and 85% in DCs. Zn2+ was either ineffective (1–10 µM) or inhibitory (40–400 µM). Recombinant rat P2X2 receptor-mediated inward currents in Xenopus oocytes displayed allosteric modulation that was different from the native guinea-pig cochlear P2X receptors. The oocyte ATP-gated inward current was potentiated 450% by shifting from pH 7.5 to pH 6.5, and 130% with 40 µM Cu2+. The enhanced response to ATP with acid pH and Cu2+ is a signature of the P2X2 subunit. In contrast to native guinea-pig cochlear cells, extracellular Zn2+ (40 µM) increased the recombinant ATP-gated inward current by 200% in oocytes. These results suggest that the positive allosteric modulation of cochlear OHC and DC ATP-gated ion channels by protons and Cu2+ arises in part from the P2X2 receptor subunit, with additional regulatory elements.