Prepulse-induced mode 2 gating behavior with and without beta-adrenergic stimulation in cardiac L-type Ca channels

Mode 2 gating of L-type Ca channels is characterized by high channel open p robability (NP0) and long openings. In cardiac myocytes, this mode is evoke d physiologically in two apparently different circumstances: membrane depol arization (prepulse facilitation) and activation of protein kinaseA. To exa mine whether the phosphorylation mechanism is involved during prepulse-indu ced facilitation of cardiac L-type Ca channels, we used isolated guinea pig ventricular myocytes to analyze depolarization-induced modal gating behavi or under different basal levels of phosphorylation. In control, NP0 measure d at 0 mV was augmented as the duration of prepulse to + 100 mV was prolong ed from 50 to 400 ms. This was due to the induction of mode 3 gating behavi or clustered at the beginning of test pulses. Analysis of open time distrib ution revealed that the prepulse evoked an extra component, the time consta nt of which is not dependent on prepulse duration. When isoproterenol (1 mu M) was applied to keep Ca channels at an enhanced level of phosphorylation , basal NP0 without prepulse was increased by a factor of 3.6 +/- 2.2 (n = 6). Under these conditions, prepulse further increased NP0 by promoting lon g openings with the same kinetics of transition to mode 2 gating (tau congr uent to 200 ms at +100 mV). Likewise, recovery fi om mode 2 gating, as esti mated by the decay of averaged unitary current, was not affected after beta -stimulation (tau congruent to 25 ms at 0 mV). The kinetic behavior indepen dent from the basal level of phosphorylation or activity of cAMP-dependent protein kinase suggests that prepulse facilitation of the cardiac Ca channe l involves a mechanism directly related to voltage-dependent conformational change rather than voltage-dependent phosphorylation.