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)=29.5+/-12 microM, 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.

Figure 1

Roscovitine slowed activation of Ca_(V)1.2 channels. (a) Ca_(V)1.2 currents were activated during 25-ms voltage steps to 30 mV. Roscovitine (Rosc; 100 μM) induced a slowing of activation and inhibition compared to currents before (Cntl) and upon recovery from (WO) roscovitine application. (b) The current–voltage (I–V) relationship shows roscovitine-induced inhibition across all current-generating voltages compared to control and washout. (c) The activation–voltage relationship was measured from tail currents and is shown normalized to maximum current to highlight the small changes induced by roscovitine (symbols are same as in b). The smooth lines are fits using a single Boltzmann function with V_1/2=7.9, 6.6 and 9.1 mV, and slope (k)=12.1, 9.9 and 12.9 for control, roscovitine and washout, respectively. (d) Activation τ (τ_Act) was generated from single exponential fits to current activation (after a 0.3-ms delay) in control, 100 μM roscovitine and washout (symbols are same as in b).

Figure 2

Roscovitine did not dissociate from Ca_(V)1.2 channels upon channel opening. (a) Ca_(V)1.2 currents were evoked by two 25-ms steps to 0 mV (prepulse (Pre) and postpulse (Post)) bracketing a +70-mV conditioning step in control, 100 μM roscovitine and washout. The interval between the conditioning step and postpulse was 10 ms. (b) Superimposed prepulse- and postpulse-evoked currents show slowed activation in the presence of 100 μM roscovitine compared to control. (c) Roscovitine (100 μM) increased activation τ (τ_Act) for both prepulse and postpulse stimulation, although these results were significantly different (mean±s.d. ^**P<0.01, n=5).

Figure 3

Roscovitine-enhanced inactivation of Ca_(V)1.2 channels. (a) Ca_(V)1.2 currents evoked by 1-s steps to 30 mV show the enhancement of inactivation by 100 μM roscovitine relative to control and washout. (b) Mean and s.d. of inactivation τ (τ_Inact) obtained from fitting a single exponential function to inactivating currents in control and 100 μM roscovitine. Data are significantly different (^**P<0.01, n=7).

Figure 4

The roscovitine-induced enhancement of inactivation saturated at high drug concentrations. (a) Representative traces from a single cell show the enhancement of inactivation induced by application of 10, 30, 100 and 300 μM roscovitine compared to control. Currents were evoked by 200-ms steps to 25 mV. (b) Inactivation was quantified as the I_End/I_Peak ratio, where I_End was measured at the end of the 200-ms step and I_Peak was measured at the peak current during the step. The smooth line is a fit using the Hill equation with an EC₅₀=29.5 μM and a Hill coefficient of 2.3. Data are presented as mean±s.d. of six cells.

Figure 5

Roscovitine slowed recovery from inactivation. (a) This set of Ca_(V)1.2 currents shows the recovery from inactivation for 10-ms (top) and 200-ms (bottom) intervals between the inactivating pulse and postpulse. Roscovitine (100 μM) increased inactivation during the 300-ms step to 50 mV and slowed recovery from inactivation at −120 mV relative to control and washout. Pre=prepulse; Post=postpulse. (b) The I_Post/I_Pre ratio is plotted vs recovery time for data from the same cell as in (a). Roscovitine (100 μM) significantly slowed the fast recovery component relative to control and washout. The smooth curves are fits using a single exponential function. (c) The mean and s.d. of recovery τ (τ_Recov) are shown for control, 100 μM roscovitine and washout. Data are significantly different (^*** P<0.001, n=4).

Figure 6

Roscovitine enhanced voltage-dependent (VDI) but not calcium-dependent inactivation (CDI). (a) The I_Post/I_Pre ratio (left axis) was measured as in Figure 5 and is plotted vs inactivation voltage to show inactivation in 10 mM Ca²⁺. Data are shown for control, 100 μM roscovitine and washout. The activation–voltage relationship in control (right axis, open circle) was measured as in Figure 1 and is superimposed here for comparison with the voltage dependence of inactivation. Data were collected in the presence of 10 mM Ca solution. (b) The voltage dependence of inactivation in 10 mM Ba²⁺ was measured as in (a). The same cell was first recorded in 10 mM Ca²⁺ (a), which was then replaced with 10 mM Ba²⁺ external solution. (c) Ca_(V)1.2 currents evoked by the triple-pulse inactivation protocol used to generate the data of (a) and (b). The 200-ms inactivation pulse to +30 mV is flanked by two 25-ms steps to 15 mV (prepulse and postpulse). Currents were recorded in 10 mM Ba²⁺ external solution in control, 100 μM roscovitine and washout. (d) 100 μM roscovitine induced a monotonic increase of inactivation with voltage in both 10 mM Ca²⁺ (n=7) and Ba²⁺ (n=5). The roscovitine-induced percent change in the I_Post/I_Pre ratio was calculated by averaging control and washout values. There was no significant difference in the roscovitine-induced percent change of inactivation between Ca²⁺ and Ba²⁺ at any voltage.

Figure 7

Closed-state inactivation was not affected by roscovitine. (a) Time course of inhibition of currents evoked by 25-ms steps to 30 mV from holding potential of −120 vs −60 mV. (b) Average fractional of inhibition by 100 μM roscovitine of currents generated by steps to 30 mV from holding potentials ranging from −120 to −60 mV (20 mV increments). Data are presented as mean±s.d. of five cells.

Figure 8

Roscovitine inhibited Ca_(V)1.2 current activated by a cardiac action potential waveform. Currents are shown for control, washout and in 30 μM roscovitine.