DIHYDROPYRIDINE-SENSITIVE SKELETAL-MUSCLE CA CHANNELS IN POLARIZED PLANAR BILAYERS .3. EFFECTS OF PHOSPHORYLATION BY PROTEIN-KINASE-C

The effects of protein kinase C (PKC) were studied on dihydropyridine (DHP)-sensitive Ca channels from rabbit skeletal muscle T tubule membranes. To determine which channel subunits become phosphorylated under the conditions used for electrophysiological studies, we first performed biochemical studies of phosphorylation. T tubular membranes were fused with vesicles of the lipid mixture used in the planar bilayers, and phosphorylation was assessed using the same concentrations of PKC, adenosine 5'-triphosphate, and buffers as were used in the electrophysiological experiments. The alpha-1 subunit of the DHP receptors was phosphorylated by PKC to an extent of 1 mol phosphate/mol protein. The beta-subunit was also phosphorylated but to a significantly lesser extent. The DHP-sensitive Ca channel activity was studied after fusing T tubule membranes with planar bilayers (Ma, J., C. Mundina-Weilenmann, M. M. Hosey, and E. Rios. 1991. Biophys. J. 60:890-901 ). The bilayers were held at -80 mV and activated by depolarizing voltage clamp pulses, The observed Ca channels exhibited two open states (tau(o1) = 5 ms and tau(o2) = 25 ms). On addition of purified PKC to the intracellular side, the proportion of the longer open state increased threefold. The average open probability during a 2-s, maximally activating pulse (P(max)BAR) increased from 10 to 15%. The voltage dependence of activation was not changed by PKC; the Boltzmann parameters were V(t) = -20.5 mV and K = 10,5 mV, which were not significantly different from the reference channels. The deactivation (closing) time constant was increased from 7 to 12 ms after PKC. The inactivation time constant during the pulse was slightly increased (from 1.2 to 1.6 s), and the channel availability at the holding potential was decreased from 76 to 71%. Taken together, the results revealed that PKC increased P(max)BAR largely through a shift in the voltage independent open-close equilibrium of the fully activated channels. This is in contrast with the eff ect of phosphorylation by PKA (Mundina-Weilenmann, C., J. Ma, E. Rios, and M. M. Hosey. 1991. Biophys. J. 60:902-909), which also increases P(max)BAR, but mostly by increasing the availability of channels and slowing inactivation during the pulse.