Characterization of voltage gated sodium channels in ovine gonadotrophs: relation to hormone secretion

Abstract

1. The properties of whole-cell Na+ currents (INa) were studied in immunocytochemically identified ovine gonadotrophs using the patch clamp technique. 2. Voltage recording under current clamp revealed that gonadotrophs did not fire spontaneously, and fired only a single action potential in response to a depolarizing current clamp step. 3. Under voltage clamp, INa was found to be sensitive to tetrodotoxin (TTX) and had an activation threshold of about -75 mV, with peak current occurring at -20 to -30 mV. 4. Using a two-pulse protocol a delay in the onset of inactivation was observed, suggesting that inactivation is dependent on and preceded by the activation phenomenon. 5. Kinetics of recovery from inactivation of the Na+ channels were studied with test pulses applied at various times after a depolarizing pre-pulse. Recovery from inactivation showed an initial delay, in contrast to the predictions of the Hodgkin-Huxley equations. 6. Recovery from inactivation was examined by using a repetitive pulse protocol, showing approximately 1 s is required for the channels to achieve a 95% recovery. 7. The steady-state inactivation (h infinity -V) curve was sigmoidal and fitted by a logistic growth curve model. The half-inactivation value of the Na+ current occurred at a membrane potential of -70 +/- 8 mV. 8. Noise power spectra derived from fluctuations of INa could be fitted with a single Lorentzian function, and the time constant value was slower at more depolarizing potentials. 9. The single-Na+-channel conductance was estimated from fluctuation analysis under conditions of reduced Na+ current amplitude by depolarizing pre-pulses. The single-channel conductance derived by the above method (approximately equal to 11 pS) corresponded to the single-channel conductance derived from single-channel current measurements using the outside-out version of the patch clamp technique (approximately equal to 13 pS). 10. Inactivation of INa was slowed by including 15 mM-iodate in the pipette. Ensemble fluctuation analysis of INa under these conditions was carried out using the steady state portion of the inactivation phase of the modified INa records, revealing a process best fitted by a double Lorentzian power spectrum, consistent with inactivation kinetics involving both a fast and a slow process. The time constant values correlated well with those obtained from a double-exponential fit to the decaying inactivation phase of the iodate-modified INa.