1. The proton-gated current was investigated in freshly dissociated ventromedial hypothalamic (VMH) neurones from 4-week-old Wistar rats, under whole-cell configuration by the use of the 'concentration-clamp' technique which combines intracellular perfusion with the rapid exchange of external solution within 1-2 ms under a single-electrode voltage-clamp condition. 2. The proton-gated current increased in a sigmoidal fashion as extracellular pH (pH(o)) decreased. In external solution containing 2 mM-Ca2+, the threshold of current activation was at pH(o) 6.5, and the maximum response appeared at pH(o) 4.1-3.9. The dissociation constant (K(d)) and Hill coefficient were 10(-4.9) M (pH(o) = 4.9) and 1.5 respectively. 3. Decreasing extracellular Na+ concentration reduced the proton-gated current. The current reversed direction at the Na+ equilibrium potential (E(Na)), indicating that it was carried by Na+. 4. The activation phase kinetics of proton-induced current was single exponential. The time constant of activation (tau(a)) did not have a potential dependence but decreased slightly by decreasing pH(o). The inactivation phase kinetics was two-exponential. The time constant of inactivation (tau(i)) consisted of fast and slow components (tau(if) and tau(is), respectively). Like tau(a), both tau(if) and tau(is) did not have any potential dependence, but they slightly increased with decreasing pH(o). 5. The steady-state inactivation curve, constructed by decreasing pH(o) from various conditioning pH(o)S to 4.1, revealed that the proton-induced current had a half-maximum inactivation at pH(o) 6.2. 6. The proton-induced current was suppressed as the extracellular Ca2+ concentration ([Ca2+]o) increased from almost free (0.01 mM) to 80 mM. Increasing [Ca2+]o increased tau(a), but slightly decreased both tau(if) and tau(is). 7. Recovery of proton-induced current from complete inactivation of proton-induced current depended on the degree of pH(o) change. A bigger change in pH(o) induced faster recovery than a smaller change. 8. External divalent cations inhibited the proton-induced current, and the inhibitory potency was in the order of Mn2+ > Co2+ > Ca2+ > Sr2+ > Ba2+ > Mg2+. 9. Tetrodotoxin (TTX) at relatively low concentration (< 10(-7) M) did not inhibit the peak amplitude of the proton-induced current, but at a higher concentration (10(-6) M) it slightly inhibited the peak amplitude of the current and accelerated the inactivation process. Scorpion toxin markedly increased the peak amplitude of the proton-induced current and prolonged the inactivation phase. The tau(is) was also increased by scorpion toxin in a concentration-dependent manner. Veratridine had no effect on the proton-induced current. 10. The membrane properties of the proton-operated channel were similar to those of the voltage-gated Na+ channel rather than the Ca2+ channel.
