title: Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 3. Effects of phosphorylation by protein kinase C.
abstract: 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. Mundiña-Weilenmann, M. M. Hosey, and E. Ríos. 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 (Pmax) increased from 10 to 15%. The voltage dependence of activation was not changed by PKC; the Boltzmann parameters were V1 = -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 Pmax largely through a shift in the voltage independent open-close equilibrium of the fully activated channels.This is in contrast with the effect of phosphorylation by PKA (Mundir'a-Weilenmann, C., J. Ma, E. Rios, and M. M. Hosey. 1991. Biophys.J. 60:902-909), which also increases Pmax but mostly by increasing the availability of channels and slowing inactivation during the pulse.
authors.affiliation: Department of Physiology, Rush University School of Medicine, Chicago, Illinois 60612.
authors.forename: J
authors.initials: J
authors.lastname: Ma
authors.forename: L M
authors.initials: LM
authors.lastname: Gutiérrez
authors.forename: M M
authors.initials: MM
authors.lastname: Hosey
authors.forename: E
authors.initials: E
authors.lastname: Ríos
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D004095:Dihydropyridines / Q000494:pharmacology*; D015640:Ion Channel Gating; D007700:Kinetics; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008564:Membrane Potentials; D009132:Muscles / Q000502:physiology*; D010766:Phosphorylation; D011336:Probability; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D013997:Time Factors
chemical_list: D015220:Calcium Channels; D004095:Dihydropyridines; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D011494:Protein Kinases; D011493:Protein Kinase C