High-voltage-activated Ca2+ channels are essential for diverse biological processes. They are composed of four or five subunits, including alpha1, alpha2-delta, beta and gamma (ref. 1). Their expression and function are critically dependent on the beta-subunit, which transports alpha1 to the surface membrane and regulates diverse channel properties. It is believed that the beta-subunit interacts with alpha1 primarily through the beta-interaction domain (BID), which binds directly to the alpha-interaction domain (AID) of alpha1; however, the molecular mechanism of the alpha1-beta interaction is largely unclear. Here we report the crystal structures of the conserved core region of beta3, alone and in complex with AID, and of beta4 alone. The structures show that the beta-subunit core contains two interacting domains: a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain. The AID binds to a hydrophobic groove in the GK domain through extensive interactions, conferring extremely high affinity between alpha1 and beta-subunits. The BID is essential both for the structural integrity of and for bridging the SH3 and GK domains, but it does not participate directly in binding alpha1. The presence of multiple protein-interacting modules in the beta-subunit opens a new dimension to its function as a multi-functional protein.

SummaryThe plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating exchange of water vapor and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. We determined the crystal structure of a bacterial homolog of SLAC1 at 1.20Å resolution, and we have used structure-inspired mutagenesis to analyze the conductance properties of SLAC1 channels. SLAC1 is a symmetric trimer composed from quasi-symmetric subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features suggest a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics suggest that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

G-protein (G␤␥)-mediated voltage-dependent inhibition of N-and P/Q-type Ca2ϩ channels contributes to presynaptic inhibition and short-term synaptic plasticity. The voltage dependence derives from the dissociation of G␤␥ from the inhibited channels, but the underlying molecular and biophysical mechanisms remain largely unclear. In this study we investigated the role in this process of Ca 2ϩ channel ␤ subunit (Ca v ␤) and a rigid ␣-helical structure between the ␣-interacting domain (AID), the primary Ca v ␤ docking site on the channel ␣ 1 subunit, and the pore-lining IS6 segment. G␤␥ inhibition of P/Q-type channels was reconstituted in giant inside-out membrane patches from Xenopus oocytes. Large populations of channels devoid of Ca v ␤ were produced by washing out a mutant Ca v ␤ with a reduced affinity for the AID. These ␤-less channels were still inhibited by G␤␥, but without any voltage dependence, indicating that Ca v ␤ is indispensable for voltage-dependent G␤␥ inhibition. A truncated Ca v ␤ containing only the AID-binding guanylate kinase (GK) domain could fully confer voltage dependence to G␤␥ inhibition. G␤␥ did not alter inactivation properties, and channels recovered from G␤␥ inhibition exhibited the same activation property as un-inhibited channels, indicating that G␤␥ does not dislodge Ca v ␤ from the inhibited channel. Furthermore, voltage-dependent G␤␥ inhibition was abolished when the rigid ␣-helix between the AID and IS6 was disrupted by insertion of multiple glycines, which also eliminated Ca v ␤ regulation of channel gating, revealing a pivotal role of this rigid ␣-helix in both processes. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the G␤␥-binding pocket through a rigid ␣-helix induced partly by the Ca v ␤ GK domain, causes the dissociation of G␤␥ and is fundamental to voltage-dependent G␤␥ inhibition.

The beta-subunit of voltage-gated Ca(2+) channels plays a dual role in chaperoning the channels to the plasma membrane and modulating their gating. It contains five distinct modular domains/regions, including the variable N- and C-terminus, a conserved Src homology 3 (SH3) domain, a conserved guanylate kinase (GK) domain, and a connecting variable and flexible HOOK region. Recent crystallographic studies revealed a highly conserved interaction between the GK domain and alpha interaction domain (AID), the high-affinity binding site in the pore-forming alpha(1) subunit. Here we show that the AID-GK domain interaction is necessary for beta-subunit-stimulated Ca(2+) channel surface expression and that the GK domain alone can carry out this function. We also examined the role of each region of all four beta-subunit subfamilies in modulating P/Q-type Ca(2+) channel gating and demonstrate that the beta-subunit functions modularly. Our results support a model that the conserved AID-GK domain interaction anchors the beta-subunit to the alpha(1) subunit, enabling alpha(1)-beta pair-specific low-affinity interactions involving the N-terminus and the HOOK region, which confer on each of the four beta-subunit subfamilies its distinctive modulatory properties.