Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder)

Abstract

Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Halt, and J. Dudel. 1987. Neurosci. Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in <1 ms. Elevation of [Ca2+]i from <10 nM to 3, 6, 20, 50, 320, or 1,000 μM activated several maxi K+ channels in the patch, whereas return to <10 nM deactivated them. The time course of Ca2+-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started with a ∼10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37°C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N·Po, on [Ca2+]i yielded a Hill coefficient of ∼3. The apparent dissociation constant, KD, decreased from 13 μM at -50 mV to 0.5 μM at +70 mV. The dependence of N·Po on voltage followed a Boltzmann distribution with a maximal Po of 0.8 and a slope factor of ∼39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.