Interplay of voltage and Ca-dependent inactivation of L-type Ca current

Abstract

Inactivation of L-type Ca channels (LTCC) is regulated by both Ca and voltage-dependent processes (CDI and VDI). To differentiate VDI and CDI, several experimental and theoretical studies have considered the inactivation of Ba current through LTCC (I(Ba)) as a measure of VDI. However, there is evidence that Ba can weakly mimic Ca, such that I(Ba) inactivation is still a mixture of CDI and VDI. To avoid this complication, some have used the monovalent cation current through LTCC (I(NS)), which can be measured when divalent cation concentrations are very low. Notably, I(NS) inactivation rate does not depend on current amplitude, and hence may reflect purely VDI. However, based on analysis of existent and new data, and modeling, we find that I(NS) can inactivate more rapidly and completely than I(Ba), especially at physiological temperature. Thus VDI that occurs during I(Ba) (or I(Ca)) must differ intrinsically from VDI during I(NS). To account for this, we have extended a previously published LTCC mathematical model of VDI and CDI into an excitation-contraction coupling model, and assessed whether and how experimental I(Ba) inactivation results (traditionally used in VDI experiments and models) could be recapitulated by modifying CDI to account for Ba-dependent inactivation. Thus, the view of a slow and incomplete I(NS) inactivation should be revised, and I(NS) inactivation is a poor measure of VDI during I(Ca) or I(Ba). This complicates VDI analysis experimentally, but raises intriguing new questions about how the molecular mechanisms of VDI differ for divalent and monovalent currents through LTCCs.

Figure 1

I_Ca inactivation: role of Ca released from the SR and of Ca permeating LTCC; I_Ba and I_NS inactivation. A. Normalized Ca, Ba and Na currents (I_Ca, I_Ba & I_NS) measured at 0 mV (except I_NS at −30 mV to obtain comparable activation state). I_Ca was recorded under conditions where normal SR Ca release and Ca transients were allowed to occur (perforated patch) or prevented (ruptured patch with cells dialyzed with 10 mM EGTA). I_Ba was also recorded in absence of Ca release and transient. I_NS was measured in divalent-free conditions (10 mM EDTA inside and out). B: Ratio between pedestal and peak current, in response to a 1000 ms- depolarizing step to 0 mV, is plotted against current amplitude of I_Ba and I_NS. The lines represent linear regressions. Data are from Brunet et al. (Brunet et al., 2009). C. I_NS and I_Ca availability through Ca channels (at −10 mV) after 500 ms pulses to the indicated E_m in guinea-pig ventricular myocytes (redrawn from (Hadley and Hume, 1987)).

Figure 2

Seven-state Markov model of I_Ca. Grey lines correspond to voltage-dependent transitions and dashed lines correspond to ion-dependent transitions. A 5-state model (5 lower states) without ion-dependent transitions was used for comparison in some simulations.

Figure 3

I_NS inactivation. A: representative recordings of membrane currents in rabbit ventricular myocytes elicited by 200-ms steps (inset) from -65 mV to the indicated E_m. B: τ of I_NS decay. C, D: I_NS availability was recorded at 25 °C and 35 °C following pre-pulse steps of 362, 148, 59, 26, 6.5, 3.5, 1.75 ms duration. Data from C and D have been transposed onto a time scale (availability against prepulse interval) and τs were calculated as in (Findlay, 2002b).

Figure 4

A: Voltage dependence of the decay time of I_NS. Data from Mitarai et al. (▸) were collected at physiological temperature. All the other experimental data were obtained at room temperature and here rescaled at 37 °C (Q₁₀=1.85). The dashed line is obtained by interpolation of the averaged τs. B: Rates of the fast (◇,□) and slow (◆,■) components of a double exponential fit of I_Ca decay vs. E_m recorded by Mahajan et al. (Mahajan et al., 2008) with and without Ca transient (CaT). Rates of I_Ba (○ and inset) and I_NS (dashed lines) inactivation are obtained from monoexponential fits of current decay.

Figure 5

I_Ba inactivation. A: Normalized I_Ba traces obtained when applying a voltage step to 0 mV (from a holding potential of -80 mV) and k_Ca/k_Ba is equal to 100, 50, 30, 20, 10, 3, 1 (VDI+f(CDI)). I_Ba was also simulated as independent of Ba ion flux (VDI only, dashed line). B: Left ordinate: τ of I_Ba decay decreases as the simulated Ba affinity for CaM is increased (i.e. k_Ca/k_Ba decreased). Right ordinate: Ion(Ba)-dependent state occupancy during I_Ba decay (in response to a voltage step to 0 mV) is enhanced with increasing affinity of Ba for CaM (i.e., decreasing k_Ca/k_Ba). C: Effect of intracellular Ba accumulation on current inactivation. Normalized I_Ba traces obtained in response to a voltage step to 0 mV are shown in correspondence of various levels of [Ba]_i (from 1 nM to 150 μM).

Figure 6

τs of I_Ba decay at the indicated E_m when I_Ba inactivation is simulated as independent of Ba ion flux (VDI only, dashed line) and when Ba affinity for CaM is varied ((VDI+f(CDI)), k_Ca/k_Ba equal to 100, 50, 30, 20, 10, 3, 1). Experimental data are from (Mahajan et al., 2008).