Effects of external protons on single cardiac sodium channels from guinea pig ventricular myocytes

Abstract

The effects of external protons on single sodium channel currents recorded from cell-attached patches on guinea pig ventricular myocytes were investigated. Extracellular protons reduce single channel current amplitude in a dose-dependent manner, consistent with a simple rapid channel block model where protons bind to a site within the channel with an apparent pKH of 5.10. The reduction in single channel current amplitude by protons is voltage independent between -70 and -20 mV. Increasing external proton concentration also shifts channel gating parameters to more positive voltages, consistent with previous macroscopic results. Similar voltage shifts are seen in the steady-state inactivation (h∞) curve, the time constant for macroscopic current inactivation (τh), and the first latency function describing channel activation. As pHo decreases from 7.4 to 5.5, the midpoint of the h∞ curve shifts from -107.6 ± 2.6 mV (mean ± SD, n = 16) to -94.3 ± 1.9 mV (n = 3, P < 0.001). These effects on channel gating are consistent with a reduction in negative surface potential due to titration of negative external surface charge. The Gouy-Chapman-Stern surface charge model incorporating specific proton binding provides an excellent fit to the dose-response curve for the shift in the midpoint of the h∞ curve with protons, yielding an estimate for total negative surface charge density of -le/490 Å2 and a pKH for proton binding of 5.16. By reducing external surface Na+ concentration, titration of negative surface charge can also quantitatively account for the reduction in single Na+ channel current amplitude, although we cannot rule out a potential role for channel block. Thus, titration by protons of a single class of negatively charged sites may account for effects on both single channel current amplitude and gating.