HEK293 cells transfected with Cav1.4α1 revealed a voltage-dependent
I Ba that was not different from
I Ba of control cells transfected with an empty vector (current densities at
V max in 30 mM Ba
2+ Ca
v1.4α1: −1.2 ± 0.2 pA/pF,
n = 12; empty vector: −1.3 ± 0.3 pA/pF,
n = 7). This finding indicated that Ca
v1.4α1 was not able to induce the formation of a functional LTCC. In contrast, when Ca
v1.4α1 was coexpressed with α2δ1 and β2a subunits, amplitudes of
I Ba consistently exceeded those of the endogenous current. In addition, the biophysical properties of the heterologously expressed current were clearly different from those of the endogenous current.
Figure 1A shows representative Ba
2+ current traces of Ca
v1.4α1 in comparison to current traces of the smooth muscle and cardiac type Ca
v1.2α1. It is evident that Ca
v1.4α1 activated faster (τ
fast: 0.65 ± 0.04 ms; τ
slow: 3.6± 0.58 ms;
n = 24; relative contribution of slow component: 0.35 ± 0.05) than Ca
v1.2α1 (τ: 1.59 ± 0.47 ms,
n = 8). Moreover, Ca
v1.4α1 displayed extremely slow inactivation kinetics. During the 150-ms voltage step shown in
Figure 1A the current did not significantly decrease. At +10 mV it took more than 30 seconds for full inactivation (not shown). Consistent with the properties of an LTCC Ca
v1.4α1 activated at relatively positive membrane potentials. In experiments performed with 30 mM Ba
2+ as the charge carrier, the mean
I–
V relationships of Ca
v1.4α1 and Ca
v1.2α1 were almost identical
(Fig. 1B) . The threshold for Ca
v1.4α1 current activation was −28 ± 1.2 mV (
n = 30), which is 5 mV more negative than Ca
v1.2α1 current activation (−23.4 ± 1.9 mV;
n = 9). For both α
1 subunits, the peak current occurred at similar
V max with 13.8 ± 0.9 mV (
n = 30) for Ca
v1.4α1 and 13.3 ± 1.7 mV (
n = 9) for Ca
v1.2α1
(Fig. 1B) . The peak current densities for Ca
v1.4α1 was −9.5 ± 1.1 pA/pF (
n = 30) and −31.4 ± 9.4 pA/pF (
n = 9) for Ca
v1.2α1. To compare the voltage-dependent activation and inactivation of the Ca
v1.4α1 and Ca
v1.2α1 currents, normalized conductance-voltage relations and steady state inactivation curves of
I Ba were determined and fitted by Bolzmann distributions
(Fig. 1C) . The activation curves for both α1 subunits were almost identical. The potential of half-maximum
I Ba activation (
V 0.5,act) was 1.1 ± 1.0 mV for Ca
v1.4α1 (
n = 28) and −0.1 ± 1.2 mV for Ca
v1.2α1 (
n = 9). In contrast, at a conditioning pulse duration of 5 seconds, the steady state inactivation curve of Ca
v1.4α1 was shifted to approximately 20 mV more depolarized potentials with respect to Ca
v1.2α1. The potential of half-maximum
I Ba inactivation (V
0.5,inact) was 0.64 ± 2.6 mV for the Ca
v1.4α1 subunit (
n = 9) and −24.3 ± 1.5 mV for the Ca
v1.2α1 subunit (
n = 9).