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

Abstract

L-type (Ca V 1.2) calcium channel antagonists play an important role in the treatment of cardiovascular disease. (R)-Roscovitine, a trisubstituted purine, has been shown to inhibit L-currents by slowing activation and enhancing inactivation. This study utilized molecular and pharmacological approaches to determine whether these effects result from (R)-roscovitine binding to a single site. Using the S enantiomer, we find that (S)-roscovitine enhances inactivation without affecting activation, which suggests multiple sites. This was further supported in studies using chimeric channels comprised of N-and L-channel domains. Those chimeras containing L-channel domains I and IV showed (R)-roscovitine-induced slowed activation like that of wild type L-channels, whereas chimeric channels containing L-channel domain I responded to (R)-roscovitine with enhanced inactivation. We conclude that (R)-roscovitine binds to distinct sites on L-type channels to slow activation and enhance inactivation. These sites appear to be unique from other calcium channel antagonist sites that reside within domains III and IV and are thus novel sites that could be exploited for future drug development. Trisubstituted purines could become a new class of drugs for the treatment of diseases related to hyperfunction of L-type channels, such as Torsades de Pointes.Cardiac L-type (Ca V 1.2) channels are central to the regulation of a number of physiological processes (1, 2). Activation of these channels in cardiac and smooth muscle generates the calcium influx that triggers calcium release from the sarcoplasmic reticulum (3) to induce contraction. In contrast, inactivation of these channels provides a negative feedback mechanism to limit calcium influx into the cardiac myocyte, which helps protect from excessive calcium influx that can lead to cardiac arrhythmias (3). L-channel antagonists are routinely used to treat cardiovascular diseases, such as hypertension and angina pectoris (4 -8). Therefore, drugs that inhibit L-channel function have high clinical relevance.Roscovitine is a 2,6,9-trisubstituted purine that was originally developed as a selective blocker of cyclin-dependent kinases (9) and is currently undergoing phase II clinical trials as an anticancer drug (10). It has recently become apparent that roscovitine can affect voltage-dependent ion channels at clinically relevant concentrations (10 -50 M) (10 -13). We (14, 15) and others (16,17) have shown that (R)-roscovitine differentially affects voltage-dependent calcium channels. (R)-Roscovitine has two effects on Ca V 2 channels (N-type, P/Q-type, and R-type), which are a rapid onset agonist effect and a more slowly developing antagonist effect (14, 15). The agonist effect results from (R)-roscovitine specifically binding to activated Ca V 2 channels to slow channel closing (14, 15), which results in a significant enhancement of action potential induced calcium influx (14). The antagonist effect appears to result from (R)-roscovitine preferentially enhancing occupancy of a "resting" inac...

Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del‐CFTR by a CDK‐independent mechanism

“…Roscovitine has also been shown to affect the activity of ionic channels though a CDK‐independent process, as in the present study for CFTR channels. For example, roscovitine binds to L‐type calcium channels (Yarotskyy et al ., ) and blocks various voltage‐dependent potassium channels (K v 1.3, K v 2.1, K v 4.3 and K v 11.1) by an open‐channel mechanism (Ganapathi et al ., ). Interestingly, unlike other hERG channel blockers, roscovitine is not trapped by hERG channel closing leading to the absence of arrhythmias (Ganapathi et al ., ).…”

Modeling Timothy Syndrome with iPS Cells

“…Basic researchers using rodent models of TS have reported that the ablation of the anchoring molecule AKAP150 and inhibition of the calmodulin-dependent protein kinase II (CaMKII) can restore normal functions in Ca V 1.2 channels(3, 4). Yarotskyy et al found that Roscovitine (Ros), a cycline-dependent kinase inhibitor, increased voltage-dependent inactivation of Ca V 1.2 channels and restored normal kinetics in TS Ca V 1.2 channels in HEK 293 cells expressing Ca V 1.2 channel constructs(31, 32). Based on this finding, we investigated whether Ros could restore normal calcium handling in TS cardiomyocytes.…”

Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome

“…The conventional L-type calcium channel blocker nimodipine failed to reverse the excess expression of TH in these neurons. Earlier studies in cardiomyocytes 12 from TS individuals indicated that roscovitine 2,3,29 , a cyclin-dependent kinase blocker that also increases L-type channel inactivation, can reduce the prolongation of the cardiac AP in cells from TS individuals. Treatment with roscovitine caused a 68% reduction in the proportion of TH + neurons (Fig.…”

“…In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type–channel blocker 2, 3, 4 . These findings provide strong evidence that Ca v 1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.…”