CYCLIC AMP-DEPENDENT PHOSPHORYLATION MODIFIES THE GATING PROPERTIES OF L-TYPE CA2+ CHANNELS IN BOVINE ADRENAL CHROMAFFIN CELLS

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca2+ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (I mM), caused a 49%, 29%, and 21 % increase in Ca2+ current (I(Ca)) amplitudes evoked by voltage steps to 0. + 10, and + 20 mV respectively (mean values from eight cells, p less-than-or-equal-to 0.05). Analysis of voltage-dependent steady-state activation (m(infinity)) curves revealed a 0.70 +/- 0.27 charge increase in the activation-gate valency (z(m)) following 8-Br-cAMP perfusion. Similar responses were observed when Ba+ was the charge carrier, where z(m) was increased by 1.33 +/- 0.34 charges (n = 8). The membrane potential for half activation (V1/2) was also significantly shifted 6 mV more negative for I(Ba) (mean, n = 8). The time course for I(Ba) (and I(Ca)) activation was well described by second-order m2 kinetics. The derived time constant for activation (tau(m)) was voltage-dependent, and the tau(m)/V relation shifted negatively after 8-Br-cAMP treatment. Ca2+ channel gating rates were derived from the tau(m) and m(infinity)2 values according to a Hodgkin-Huxley type m2 activation process. The forward rate (alpha(m)) for channel activation was increased by 8-Br-cAMP at membrane potentials greater-than-or-equal-to 0 mV, and the backward rate (beta(m)) decreased at potentials less-than-or-equal-to + 10 mV. Time-dependent inactivation of I(Ca) consisted of a slowly decaying component (tau(h) almost-equal-to 300 ms) and a "non-inactivating" steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.