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pubmed:abstractText |
1. ATP-evoked currents and Ca2+ signals were simultaneously recorded in isolated inner hair cells (IHC) of guinea-pig cochlea by combining conventional whole-cell or perforated patch clamp recording with indo-1 dual emission microfluorometry. 2. In most IHCs, voltage clamped near resting membrane potential (-40 mV), extracellular ATP evoked a rapid inward current (time constant, 150 ms). This current was concomitant with a slow rise in [Ca2+]i (time constant, 5 s). The ATP-evoked inward currents could be repeated several times with only a small run-down in amplitude (< 10%), while the ATP-evoked Ca2+ responses showed a rapid run-down (> 80% at the third ATP application). 3. The current-voltage relationship of ATP-evoked currents showed a reversal potential at -11 +/- 6 mV (n = 8), suggesting that ATP essentially activated a non-specific cationic conductance. On the contrary, the amplitude of the ATP-evoked Ca2+ responses did not show significant dependence on holding membrane potential. 4. The Ca2+ response showed an apparent Kd for ATP (EC50, 1.8 +/- 0.3 microM; Hill coefficient, 1.0 +/- 0.1) eightfold smaller than for the evoked currents (EC50, 13.7 +/- 3.0 microM; Hill coefficient, 2.0 +/- 0.7). 5. Perfusion with high extracellular Ca2+ solution (10 mM CaCl2) reduced the amplitude of the ATP-evoked currents by 90%, while perfusion with zero Ca2+ solution increased it by more than 100%. However, similar variations in external Ca2+ concentration did not change the amplitude of the ATP-evoked Ca2+ responses. Furthermore, intracellular heparin (1 mg mL-1), a potent inhibitor of InsP3 receptors, did not significantly change the amplitude of ATP-evoked currents but reduced the ATP-evoked Ca2+ response, suggesting again that the latter is related to Ca2+ release from intracellular stores. 6. The results suggested that two types of P2-purinergic receptor are expressed in IHCs: ATP-gated ion channels and ATP-activated metabotropic receptors. At submicromolar ATP concentrations, the metabotropic receptors raising intracellular [Ca2+] would hyperpolarize IHCs via Ca(2+)-sensitive K+ channels. The ATP-gated ion channels activated at higher ATP concentrations would mainly have a depolarizing effect on IHCs.
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