Slowed N-Type Calcium Channel (CaV2.2) Deactivation by the Cyclin-Dependent Kinase Inhibitor Roscovitine

Abstract

The lack of a calcium channel agonist (e.g., BayK8644) for CaV2 channels has impeded their investigation. Roscovitine, a potent inhibitor of cyclin-dependent kinases 1, 2, and 5, has recently been reported to slow the deactivation of P/Q-type calcium channels (CaV2.1). We show that roscovitine also slows deactivation (EC(50) approximately 53 microM) of N-type calcium channels (CaV2.2) and investigate gating alterations induced by roscovitine. The onset of slowed deactivation was rapid ( approximately 2 s), which contrasts with a slower effect of roscovitine to inhibit N-current (EC(50) approximately 300 microM). Slow deactivation was specific to roscovitine, since it could not be induced by a closely related cyclin-dependent kinase inhibitor, olomoucine (300 microM). Intracellularly applied roscovitine failed to slow deactivation, which implies an extracellular binding site. The roscovitine-induced slow deactivation was accompanied by a slight left shift in the activation-voltage relationship, slower activation at negative potentials, and increased inactivation. Additional data showed that roscovitine preferentially binds to the open channel to slow deactivation. A model where roscovitine reduced a backward rate constant between two open states was able to reproduce the effect of roscovitine on both activation and deactivation.

Domain III regulates N-type (CaV2.2) calcium channel closing kinetics

“…The difference in Act V e between N-type and L-type channels was not correlated with any domains and thus appears to be a more distributed function. We and others have previously demonstrated that roscovitine slows deactivation of P/Q-, N-, and R-type calcium channels, which results in elevated action potential-induced calcium influx (Buraei et al 2005(Buraei et al , 2007Cho and Meriney 2006;Yan et al 2002). We show here that N-DIII is both necessary and sufficient to mediate this effect.…”

“…The tail current amplitude was plotted versus step voltage and fitted by a Boltzmann function, which yielded half-activation voltage (V 1/2 ) and slope factor. Activation and deactivation kinetics were determined with single-exponential fitting as previously described (Buraei et al 2005). Group data are calculated as means Ϯ SD throughout this report.…”

“…Thus roscovitine provides us with another method to probe structures that control N-channel closing. The requirement of Ca V 2 channel activation for the roscovitine-induced slowed deactivation, as well as the fact that externally but not internally applied roscovitine induced the effect, suggested an externally oriented binding site (Buraei et al 2005(Buraei et al , 2007. While we had previously proposed that the binding site resided on the ␣ 1B -subunit, the associated ␣ 2 ␦-subunit is extracellularly exposed, and is thought to be the target for clinically relevant compounds such as gabapentin to affect channel activity (Brown and Gee 1998).…”

“…However, roscovitine, a trisubstituted purine, can slow deactivation of Ca V 2 channels by stabilizing high-P o open state (Buraei et al 2005(Buraei et al , 2007. This effect was accompanied by an increase in the Deact V e , left shift in the activation vs. voltage relationship (⌬V 1/2 ) (Buraei et al 2005(Buraei et al , 2007, and slowed gating charge relaxation . Thus roscovitine provides us with another method to probe structures that control N-channel closing.…”

“…For wild-type (wt) N-and L-channels, both activation and deactivation time constants () were different at certain voltages, but differences at single voltages are highly susceptible to many factors that make them unreliable for our purposes. For this reason, we compared these values over a range of voltages to determine the e-fold change with voltage (V e ) (Buraei et al 2005). The V e for both activation and deactivation was significantly smaller (i.e., steeper voltage dependence) for wt N-type vs. L-type channels (Fig.…”