Modulation of slow inactivation in class A Ca2+ channels by beta-subunits

Abstract

beta-subunit modulation of slow inactivation of class A calcium (Ca2+) channels was studied with two-microlectrode voltage clamp after expression of the alpha1A- (BI-2) together with beta1a-, beta2a-, beta3- or beta4-subunits in Xenopus oocytes. On- and off-rates of slow inactivation were estimated from the kinetics of recovery from slow inactivation. Ca2+ channels with an alpha1A/beta-subunit composition inducing the slower rate of fast inactivation displayed the faster rate of slow inactivation. The corresponding order of slow inactivation time constants (tau[onset]) was: alpha1A/beta2a, 33 +/- 3 s; alpha1A/beta4, 42 +/- 4 s; alpha1A/beta1a, 59 +/- 4 s; alpha1A/beta3, 67 +/- 5 s (n >= 7). Recovery of class A Ca2+ channels from slow inactivation was voltage dependent and accelerated at hyperpolarized voltages. At a given holding potential recovery kinetics were not significantly modulated by different beta-subunits. Two mutations in segment IIIS6 (IF1612/1613AA) slowed fast inactivation and accelerated the onset of slow inactivation in the resulting mutant (alpha1A/IF-AA/beta3) in a similar manner as coexpression of the beta2a-subunit. Recovery from slow inactivation was slightly slowed in the double mutant. Our data suggest that class A Ca2+ channels enter the 'slow inactivated' state more willingly from the open than from the 'fast inactivated' state. The rate of slow inactivation is, therefore, indirectly modulated by different beta-subunits. Fast and slow inactivation in class A Ca2+ channels appears to represent structurally independent conformational changes. Fast inactivation is not a prerequisite for slow inactivation.

Figure 1. Inactivation and recovery kinetics of class A Ca²⁺ channels with different β-subunit composition

A, I_Ba through α_1A/β_1a, α_1A/β_2a, α_1A/β₃ and α_1A/β₄ channels during a 3 s depolarizing step from −80 to 10 mV. Superimposed current traces were scaled to illustrate the different inactivation properties. The I_Ba decay of α_1A/β_1a, α_1A/β₃ and α_1A/β₄ channels is dominated by fast inactivation; the α_1A/β_2a channel exhibits slow inactivation kinetics (see also Stea et al. 1994). B, recovery of α_1A/β₃ Ca²⁺ channels reveals two components in inactivation. Normalized peak currents are plotted as a function of time (see pulse protocol in the top panel). The smooth line illustrates the two components in I_Ba recovery corresponding to recovery from fast (τ_fast) and slow (τ_slow) inactivation (for estimation of τ_fast and τ_slow see Fig. 4).

Figure 4. Voltage dependence of recovery from fast and slow inactivation of class A Ca²⁺ channels with different β-subunit composition

A and B, I_Ba recovery from inactivation after 10 s conditioning pulses to 10 mV measured by a conventional double pulse protocol (similar to Fig. 1B, top panel). The recovery potentials were −120 mV (•), −100 mV (♦), −80 mV (▪) or −60 mV (▴). The holding potential was −120 mV in all experiments. Mean recovery time courses of α_1A/β₄ and α_1A/β_2a channels are shown. Data points are fitted by a biexponential function. Fit parameters are displayed in Table 1. C, time constants of recovery from fast inactivation of class A Ca²⁺ channels with different β-subunit composition at −120 mV (□), −100 mV () −80 mV (▪) and −60 mV (▪). D, time constants of recovery from slow inactivation at −120 mV (□), −80 mV (▪) and −60 mV () were estimated as described in Fig. 2A (n > 20). No significant differences with τ_slow estimated from double pulse experiments (Table 1) were observed (P > 0.05). The mean values for different subunit combinations at the corresponding holding potentials were not significantly different (P > 0.05).

Figure 2. Estimation of slow inactivation in class A Ca²⁺ channels by back extrapolation of I_Ba recovery kinetics

A, recovery from slow inactivation was observed at a holding potential of −80 mV during eight 60 ms test pulses to 10 mV applied 5, 10, 20, 30, 60, 90, 120 and 180 s after conditioning pulses of either 3, 7, 10, 20 or 30 s. Channels recovered completely from fast inactivation during the 5 s rest between the end of the conditioning pulse and the first test pulse (see Fig. 4C for τ_fast at 80 mV). Current amplitudes were normalized to I_Ba of the last test pulse and recovery kinetics fitted by an exponential function: I_N = 1 –I_S exp(-t/τ_slow), where I_N represents the normalized current amplitude, t the recovery time and τ_slow the time constant of recovery from slow inactivation. τ_slow was individually estimated for each prepulse length; no significant differences between the mean values for different prepulse lengths were observed. Extrapolation of the mono-exponential recovery to the end of the conditioning pulse (corresponding to time zero) enables the determination of the fraction of channels that have not entered the ‘slow inactivated’ state (1 –I_S) during the conditioning pulse. The inset illustrates superimposed 60 ms test pulse currents during recovery from slow inactivation after 3 or 30 s conditioning pulses. B, fractions of non-slow-inactivated channels (1 –I_S) after conditioning pulses of different lengths are plotted on a logarithmic scale. The kinetics were fitted by a linear function with the slope K_S representing the on-rate of slow inactivation. The coefficient C (y-intercept) was a free parameter of the fit (see Appendix for details).

Figure 3. Onset of slow inactivation is modulated by β-subunit composition

A, slow inactivation was estimated by using conditioning pulses of different length (Fig. 2A). The rate constants were calculated as described in Fig. 2B. A comparison of I_Ba inactivation in α_1A/β_2a and α_1A/β₃ channels during a 30 s test pulse from −80 to 10 mV is illustrated in the inset. B, on-rate constants of slow inactivation of the four different α_1A/β-subunit combinations at 10 mV. The corresponding time constants (τ_onset = 1/K_S) are α_1A/β_1a: 59 ± 4 s, n = 7; α_1A/β_2a: 33 ± 3 s, n = 12; α_1A/β₃: 67 ± 5 s, n = 10; α_1A/β₄: 42 ± 4 s, n = 7.

Figure 5. Point mutations in segment IIIS6 of α_1A (IF1612,1613AA) affect the on-rate of slow inactivation and recovery

A, onset of slow inactivation in α_1A/β₃ and α_1A/IF-AA/β₃ channels (estimated as described in Fig. 2). Slow inactivation time constants (τ_onset) of α_1A/β₃ (67 ± 5 s, n = 10) and α_1A/IF-AA/β₃ channels (38 ± 5 s, n = 6) were significantly different (P < 0.01). Representative normalized currents of both subunit combinations during a 3 s pulse from −80 mV to 10 mV are illustrated in the inset. Note the slower fast-inactivation rate in the double mutant α_1A/IF-AA/β₃. B, recovery of α_1A/β₃ and α_1A/IF-AA/β₃ channels from the slow inactivated state after 30 s conditioning pulse to +10 mV (holding potential −80 mV). Recovery time constants (τ_slow) of α_1A/β₃ (19.2 ± 0.6 s, n = 47) and α_1A/IF-AA/β₃ channels (23.8 ± 0.8 s, n = 26) were significantly different (P < 0.01).