Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder).
Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 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 degrees 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.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 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.
Markwardt
F
F
Department of Physiology, University of Cologne, Germany.
Isenberg
G
G
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004553:Electric Conductivity; D006168:Guinea Pigs; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D009130:Muscle, Smooth / Q000737:chemistry / Q000166:cytology* / Q000502:physiology; D013997:Time Factors; D001743:Urinary Bladder / Q000737:chemistry / Q000166:cytology* / Q000502:physiology
D015220:Calcium Channels; D002118:Calcium