Roscovitine, a cyclin‐dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L‐type (Ca_(V)1.2) calcium channels

Abstract

Background and purpose: L-type calcium channels (Ca (V) 1.2) play an important role in cardiac contraction. Roscovitine, a cyclin-dependent kinase inhibitor and promising anticancer drug, has been shown to affect Ca (V) 1.2 by inhibiting current amplitude and slowing activation. This research investigates the mechanism by which roscovitine inhibits Ca (V) 1.2 channels. Experimental approach: Ca (V) 1.2 channels were transfected into HEK 293 cells, using the calcium phosphate precipitation method, and currents were measured using the whole-cell patch clamp technique. Key results: Roscovitine slows activation at all voltages, which precludes one previously proposed mechanism. In addition, roscovitine enhances voltage-dependent, but not calcium-dependent inactivation. This enhancement resulted from both an acceleration of inactivation and a slowing of the recovery from inactivation. Internally applied roscovitine failed to affect Ca (V) 1.2 currents, which supports a kinase-independent mechanism and extracellular binding site. Unlike the dihydropyridines, closed state inactivation was not affected by roscovitine. Inactivation was enhanced in a dose-dependent manner with an IC 50 ¼ 29.5712 mM, which is close to that for slow activation and inhibition. Conclusions and implications: We conclude that roscovitine binds to an extracellular site on Ca (V) 1.2 channels to inhibit current by both slowing activation and enhancing inactivation. Purine-based drugs could become a new option for treatment of diseases that benefit from L-channel inhibition such as cardiac arrhythmias and hypertension.

Roscovitine Binds to Novel L-channel (CaV1.2) Sites That Separately Affect Activation and Inactivation

“…These two effects were characterized by approximately equal EC 50 values (ϳ30 M), which suggested a single binding site. However, the Hill coefficient for (R)-roscovitine-induced slowed activation was ϳ1, whereas that for enhanced inactivation was Ͼ2, which could result from multiple binding sites (19). Intracellularly applied (R)-roscovitine failed to affect L-channel activity, which supported an extracellularly exposed binding site(s) mediating both effects.…”

“…The external recording solution contained 30 mM BaCl 2 , 100 mM NMG-Cl, 10 mM NMG-HEPES, osmolarity of 300 mosM, and pH 7.3. We used Ba 2ϩ as the permeant ion to exclude Ca 2ϩ -dependent inactivation (25,26), since we have previously demonstrated that (R)-roscovitine enhances VDI but does not affect calciumdependent inactivation (19). Both (R)-and (S)-roscovitine were prepared as a 50 mM stock solution in DMSO and stored at Ϫ30°C.…”

“…HEK Cell Transfection-We utilized either the calcium phosphate precipitation method (19) or Lipofectamine 2000 (following the manufacturer's directions) to transfect HEK 293 cells with WT L-, N-, and chimeric channels, which provided highly reproducible expression 24 -72 h after transfection. HEK293 cells were maintained in standard Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic mixtures (regular medium) at 37°C in a 5% CO 2 incubator.…”

“…have shown that (R)-roscovitine significantly slows activation and enhances inactivation of L-type channels and hypothesized that a single extracellular binding site mediated both effects (19). We further investigated this idea by examining the effect of (S)-roscovitine on L-type channels, which was useful in differentiating multiple roscovitine binding sites on N-type channels (15,17,18).…”

“…L-type channels are also inhibited by (R)-roscovitine, but by a unique mechanism relative to N-channels (19). L-channel inhibition results from slowed activation and enhanced open state voltage-dependent inactivation (VDI), 3 but the resting inactivated state is not affected.…”