A single CaVβ can reconstitute both trafficking and macroscopic conductance of voltage-dependent calcium channels

High-voltage-activated (HVA) calcium channels are hetero-multimeric protein complexes comprised minimally of a pore-forming α1-subunit (CaV1.1–1.4; CaV2.1–2.3), and auxiliary β (CaVβ1–CaVβ4), α2δ (α2δ-1–α2δ-3), and sometimes γ subunits (Catterall, 2000). These proteins control Ca2+ fluxes that underlie critical biological functions ranging from muscle contraction to hormone and neurotransmitter release. Auxiliary CaVβ subunits play a crucial role in the functional maturation of HVA calcium channels: each of the four CaVβs can interact promiscuously with any of the seven known HVA calcium-channel α1 subunits to promote membrane trafficking of the associated α1 (Chien et al. 1995; Brice et al. 1997; Yamaguchi et al. 2000; Colecraft et al. 2002; Takahashi et al. 2004), to increase channel Po (Singer et al. 1991; Costantin et al. 1998; Hullin et al. 2003; Takahashi et al. 2004), and to produce hyperpolarizing shifts in the voltage-dependence of channel activation (Singer et al. 1991; Perez-Reyes et al. 1992; De Waard et al. 1994; Jones et al. 1998). The powerful impact of CaVβs on HVA calcium-channel operation logically focuses attention on the interaction between α1 and CaVβ subunits as a promising locus to modify calcium-channel behaviour. Indeed, this interaction is the target for physiological down-regulation of calcium currents by the Rem, Rad, Kir/Gem (RGK) family of Ras-like GTPases (Beguin et al. 2001; Finlin et al. 2003), and serves as the basis for high-throughput identification of novel pharmacological inhibitors of HVA calcium-channel activity (Young et al. 1998). A deeper mechanistic understanding of the HVA α1–CaVβ interaction is important for both appreciating its physiological ramifications, as well as potentially hastening the discovery of new calcium-channel therapeutics. A fundamental ambiguity of HVA calcium-channel structure relates to the functional stoichiometry of α1 to CaVβ (Birnbaumer et al. 1998; Canti et al. 2001; Jones, 2002; Dolphin, 2003). Specifically, it is unclear whether the fully functional channel is comprised of an α1 subunit associated with single or multiple CaVβ subunits. Biochemical studies initially established that α1 and CaVβ subunits copurify in an equimolar ratio (Witcher et al. 1993); the uncertainty arises from studies which indicate that trafficking and gating-modulation are separable functions dependent on distinct CaVβ concentrations. In Xenopus oocytes, expression of α1 without exogenous CaVβ leads to recovery of low-amplitude currents that activate at relatively depolarized potentials, compared to channels obtained by coexpressing α1 and CaVβ (Singer et al. 1991; Perez-Reyes et al. 1992; Birnbaumer et al. 1998). However, the presumed α1-alone currents are abolished by antisense oligonucleotides against a subsequently identified endogenous Xenopus CaVβ (CaVβXO) (Tareilus et al. 1997). These results suggested that there is sufficient endogenous CaVβXO in the Xenopus expression system to fully traffick α1 subunits to the plasma membrane, but not to modulate gating (Tareilus et al. 1997; Birnbaumer et al. 1998). In agreement, careful titration of the amount of CaVβ3 coexpressed with CaV2.2 in Xenopus oocytes indicated that the concentration of CaVβ3 required to traffick channels to the plasma membrane is nearly an order of magnitude less than that required to modulate channel gating (Canti et al. 2001). Furthermore, acute application of purified CaVβ protein was found to modulate the gating of pre-existing calcium channels in the plasma membrane (Yamaguchi et al. 1998; Garcia et al. 2002). These data are consistent with two mutually exclusive interpretations of the functional stoichiometry of the α1–CaVβ interaction (Birnbaumer et al. 1998; Jones, 2002; Dolphin, 2003). In the ‘single-CaVβ-binding’ model, CaVβ binds to α1 at the endoplasmic reticulum (ER) to promote translocation to the plasma membrane. At low CaVβ concentrations, reversible unbinding of CaVβ from α1 favours a significant steady-state fraction of low-activity CaVβ-less channels in the plasma membrane. At high CaVβ concentrations, the equilibrium is shifted towards high-activity (CaVβ-bound) channels (Fig. 1A). Thus, a single CaVβ is responsible for both trafficking and enhancing the channel Po. Alternatively, in the ‘multiple-CaVβ-binding’ model, trafficking and gating-modulation are mediated by distinct CaVβ subunits (Fig. 1B). As before, a CaVβ binds to an α1 to promote channel trafficking. After insertion into the plasma membrane, the high-affinity interaction between α1 and CaVβ persists. By contrast to the single-CaVβ model, these α1+ CaVβ channels exhibit low gating activity, and binding of a second CaVβ to a distinct lower affinity site is required for high gating activity. Distinguishing between these two models is fundamental to mechanistic understanding of CaVβ function, and has important implications for rational manipulation of the α1–CaVβ interaction to regulate calcium-channel activity. Figure 1 Alternative models proposed for the functional stoichiometry of high-voltage-activated calcium-channel α1–β subunit interaction A key obstacle to discriminating between the two models is that in traditional coexpression studies it is practically impossible to distinguish the precise stoichiometry of α1 and CaVβ subunits present in functional channels. Here, we take the novel approach of covalently linking an α1C subunit (CaV1.2) to a CaVβ2b subunit, thus ensuring a 1: 1 stoichiometry in transfected HEK 293 cells. We find that a single CaVβ is necessary and sufficient to completely reconstitute both channel trafficking and macroscopic conductance. We conclude that the single-CaVβ model captures the functional essence of HVA calcium channels.