Site and mechanism of activation of proton-induced sodium current in chick dorsal root ganglion neurones

Abstract

1. In dissociated and cultured 1- to 2-day-old chick dorsal root ganglion cells, and in isolated outside-out membrane patches, a large transient current lasting 1-2 s could be activated upon step increases in [H+]o. The proton-induced current reversed direction at the Na+ equilibrium potential, was abolished completely in the absence of Na+, and was therefore labelled INa(H). 2. To investigate the activation and deactivation kinetics of INa(H) at the single-channel level, we employed isolated membrane patches and a method whereby we could change the external solution in less than 1 ms. 3. In outside-out membrane patches, INa(H) was fully activated within 2 ms between pH 6.7 and 5.7. Half-times of activation decreased with increasing [H+]o. The calculated association rate constant was 9.5 x 10(9) M-1 s-1. 4. Deactivation of INa(H), following a step reduction in [H+]o, occurred with half-times of within 1.3-2 ms. 5. In the continued presence of an activating solution (pH 6.7 and 1 mM-Ca2+), INa(H) inactivated slowly, with a time constant of about 300 ms. 6. Inactivation showed a limited dependence on [Ca2+]o. The time constant of inactivation increased from about 300-500 ms as [Ca2+]o was decreased from 5 to 0.1 mM. Further decrease in [Ca2+]o did not significantly increase the time course of inactivation. Increases in [Ca2+]i from 10(-9) to 10(-3) M had no effect on the activation or inactivation kinetics of INa(H). 7. Conditioning proton concentrations which by themselves failed to activate any channel openings, partially inactivated INa(H). 8. Recovery from inactivation appeared to follow a time course similar to that of inactivation itself. 9. INa(H) could not be activated in inside-out patches. A step increase in proton concentration outside a cell-attached patch was also ineffective at producing INa(H) in the patch. Intracellular pH between 7.9 and 6.7 had no effect on the activation or inactivation of INa(H). 10. The activation and inactivation kinetics were not significantly voltage dependent. 11. The single-channel conductance associated with the activation of INa(H) was 28 pS in symmetrical 120 mM-NaCl solutions and remained constant throughout the time course of INa(H). 12. During activation of the voltage-gated calcium current, ICa, a step increase in proton concentration caused a rapid (ca. 2 ms) suppression of ICa which was more than that predicted from the steady-state effects of H+ on ICa. This effect was independent of [Na+] and the direction of INa(H).(ABSTRACT TRUNCATED AT 400 WORDS)