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

Ca2+ entry through voltage-gated Ca2+ channels modulates a variety of neuronal functions such as: release of neurotransmitters, generation and propagation of action potentials and gene expression. Nine classes of voltage-gated Ca2+ channels that are coded by at least nine different genes (classes A, B, C, D, E, F, G, H, I) have been identified in neuronal cells (Birnbaumer et al. 1994; Perez-Reyes et al. 1999; Lee et al. 1999). A hallmark of class A Ca2+ channels is their sensitivity to the funnel web spider venom ω-Aga-IVA (Mintz et al. 1992b). This channel type is widely distributed in the central and peripheral nervous system (Mintz et al. 1992a, b; Stea et al. 1994; Westenbroek et al. 1995) and there is evidence that Ca2+ entry through the α1A-subunit mediates the release of neurotransmitters more efficiently than other neuronal Ca2+ channels (Wu et al. 1999). Class A Ca2+ channels are hetero-oligomeric protein complexes consisting of a pore-forming α1A-subunit, at least one of four β-subunits (β1-β4) and an α2δ-subunit. The auxiliary β-subunits modulate expression density as well as voltage dependence of channel activation and inactivation kinetics (Stea et al. 1994; Olcese et al. 1994; De Waard & Campbell, 1995). Multiple β-subunits are associated with the α1A-subunit to different extents in different parts of the mammalian brain (Liu et al. 1996; Pichler et al. 1997) suggesting that class A Ca2+ channel properties are modulated by tissue-specific expression of different β-subunits (Tanaka et al. 1995). Missense mutations in α1A are associated with the aetiology of familial hemiplegic migraine (Ophoff et al. 1996; Kraus et al. 1998; Hans et al. 1999), ataxia (Zhuchenko et al. 1997) and epilepsy (Fletcher et al. 1996). Ca2+ influx through class A channels and the sensitivities to the ω-agatoxin IVA are modulated by alternative splicing of the α1A-subunit (see splice variants α1A-a and α1A-b in Bourinet et al. 1999). Class A Ca2+ channel currents decay under voltage clamp with a biexponential time course suggesting two mechanisms of inactivation. Fast inactivation (corresponding to the transient current decay) is affected by point mutations in different parts of the α1A-subunit (see Hering et al. 1998 for review; Bourinet et al. 1999). The additional slow inactivation is much less understood. We have, therefore, expressed a neuronal α1A-subunit (BI-2, Mori et al. 1991) together with β1a-, β2a-, β3- or β4-subunits in Xenopus oocytes and analysed the individual effects of various β-subunits on slow inactivation. We report here that a reduction of fast inactivation by coexpression of α1A- with the β2a-subunit simultaneously accelerates the channel state transitions into the slow inactivated state. Alternatively, slowing fast inactivation of α1A/β3 channels by mutating two inactivation determinants in segment IIIS6 to alanine (IF1612/1613AA) accelerated the onset of slow inactivation in a similar manner. Our data suggest that open class A Ca2+ channels are more willing to enter the slow inactivated state than channels in the fast inactivated conformation. The tissue-specific expression patterns of different β-subunits appear, therefore, as an indirect determinant of slow inactivation in class A Ca2+ channels.