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

1. beta-subunit modulation of slow inactivation of class A calcium (Ca2+) channels was studied with two-microlectrode voltage clamp after expression of the alpha(1A)- (BI-2) together with beta(1a)-, beta(2a)-, beta(3)- or beta(4)-subunits in Xenopus oocytes. 2. On- and off-rates of slow inactivation were estimated from the kinetics of recovery from slow inactivation. Ca2+ channels with an alpha(1A)/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: alpha(1A)/beta(2a), 33 +/- 3 s; alpha(1A)/beta(4), 42 +/- 4 s; alpha(1A)/beta(1a), 59 +/- 4 s; alpha(1A)/beta(3), 67 +/- 5 s (n greater than or equal to 7). 3. 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. 4. Two mutations in segment IIIS6 (IF1612/1613AA) slowed fast inactivation and accelerated the onset of slow inactivation in the resulting mutant (alpha(1A/IF-AA)/beta(3)) in a similar manner as coexpression of the beta(2a)-subunit. Recovery from slow inactivation was slightly slowed in the double mutant. 5. 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 Blow inactivation is, therefore, indirectly modulated by different beta-subunits. 6. 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.