Effects of external Rb+ on inward rectifier K+ channels of bovine pulmonary artery endothelial cells

Abstract

Inward rectifier (IR) K+ channels of bovine pulmonary artery endothelial cells were studied using the whole-cell, cell-attached, and outside-out patch-clamp configurations. The effects of Rb+ on the voltage dependence and kinetics of IR gating were explored, with [Rb+](o) + [K+](o) = 160 mM. Partial substitution of Rb+ for K+ resulted in voltage-dependent reduction of inward currents, consistent with Rb+ being a weakly permeant blocker of the IR. In cells studied with a K+-free pipette solution, external Rb+ reduced inward IR currents to a similar extent at large negative potentials but block at more positive potentials was enhanced. In outside-out patches, the single-channel ι-V relationship was approximately linear in symmetrical K+, but rectified strongly outwardly in high [Rb+](o) due to a reduced conductance for inward current. The permeability of Rb+ based on reversal potential, V(rev), was 0.45 that of K+, whereas the Rb+ conductance was much lower, 0.034 that of K+, measured at V(rev)-80 mV. The steady state voltage-dependence of IR gating was determined in Rb+-containing solutions by applying variable prepulses, followed by a test pulse to a potential at which outward current deactivation was observed. As [Rb+](o) was increased, the half-activation potential, V 1/2 , changed less than V(rev). In high [K+](o) solutions V 1/2 was V(rev)-6 mV, while in high [Rb+](o) V 1/2 was V(rev) + 7 mV. This behavior contrasts with the classical parallel shift of V 1/2 with V(rev) in K+ solutions. Steady state IR gating was less steeply voltage-dependent in high [Rb+](o) than in K+ solutions, with Boltzmann slope factors of 6.4 and 4.4 mV, respectively. Rb+ decreased (slowed) both activation and deactivation rate constants defined at V 1/2 , and decreased the steepness of the voltage dependence of the activation rate constant by 42%. Deactivation of IR channels in outside-out patches was also slowed by Rb+. In summary, Rb+ can replace K+ in setting the voltage-dependence of IR gating, but in doing so alters the kinetics.