Protein kinase C activation inhibits α1D L-type Ca channel:: A single-channel analysis

The recently reported alpha(1D) Ca channel in the heart is known to be regulated by protein kinase C (PKC) at the whole cell level and has been implicated in atrial fibrillation. The biophysical basis of this regulation at the single-channel level is not known. Therefore, the effect of PKC activation was studied on alpha(1D) Ca channel expressed in tsA201 cells using cell-attached configuration. Unitary currents were recorded in the presence of 70 mM Ba2+ as the charge carrier at room temperature. Under basal condition, channel activity was rare and infrequent; however, Bay K 8644 (1 mu M) induced channel openings with a conductance of 22.3 pS. Single channel analysis of open and closed time distributions were best fitted with a single exponential. PKC activation by 4 alpha-phorbol 12-myristate 13-acetate (PMA; 10 nM), a phorbol ester derivative, resulted in a decrease in open probability and increase in closed-time without any significant effect on the conductance of the alpha(1D) Ca channel. This is consistent with a decreased entry of alpha(1D) Ca channel into open states in the presence of PMA. PMA effects could not be reproduced by 4-alpha Phorbol, an inactive PMA analogue. These data show, for the first time, (1) the alpha(1D) Ca channel activity at the single-channel level and (2) the biophysical basis by which PKC activation inhibits the alpha(1D) Ca channel. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the previously reported whole-cell alpha(1D) Ca current. Altogether, these data are essential for understanding the complex role of alpha(1D) Ca channel not only in physiological settings but also in pathological settings such as atrial fibrillation.