No abstract

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.

All auxiliary ␣2␦ subunits of voltage-gated Ca 2؉ (CaV) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in ␣2␦-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the ␣2␦-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant ␣2␦-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT ␣2␦-2, the MIDAS mutant ␣2␦-2 subunit did not enhance and, in some cases, further diminished Ca V1.2, -2.1, and -2.2 currents coexpressed with ␤1b by using either Ba 2؉ or Na ؉ as a permeant ion. Furthermore, expression of the MIDAS mutant ␣2␦-2 reduced surface expression and strongly increased the perinuclear retention of Ca V␣1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108 -15 cells. Despite the presence of endogenous ␣2␦ subunits, heterologous expression of ␣2␦-2 in differentiated NG108 -15 cells further enhanced the endogenous high-threshold Ca 2؉ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that ␣2␦ subunits normally interact with the CaV␣1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the ␣2␦ subunit is required to promote trafficking of the ␣1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains.integrin ͉ neuron ͉ motif ͉ expression V oltage-gated Ca 2ϩ (Ca V ) channels are composed of a poreforming ␣1 subunit that determines the main biophysical properties of the channel. For the Ca V 1 and -2 subfamilies, this subunit is associated with an intracellular ␤ subunit (for review, see refs. 1 and 2) and a membrane-anchored, predominantly extracellular ␣ 2 ␦ subunit (for review, see ref.3). Mammalian genes encoding 10 ␣1, 4 ␤, and 4 ␣ 2 ␦ subunits have been identified (for reviews, see refs. 2 and 4). The topology of the ␣ 2 ␦ protein has been determined in detail only for ␣ 2 ␦-1 but is thought to generalize to all 4 ␣ 2 ␦ subunits (for review, see ref.3). All ␣ 2 ␦ subunits have predicted N-terminal signal sequences, indicating that the N terminus is extracellular. In early studies of ␣ 2 ␦-1 purified from skeletal and cardiac muscle, it was determined that the ␣ 2 subunit is disulfide-bonded to a transmembrane ␦ subunit, and both subunits are the products of a single gene, encoding the ␣ 2 ␦ protein, that is posttranslationally cleaved into ␣ 2 and ␦ (5).Subsequent to the identification of ␣ 2 ␦ subunits as stoichiometric components of skeletal muscle Ca 2ϩ channels, ␣ 2 ␦ subunits have also been shown to be associated with native cardiac (L-type) (6) and neuronal N-and P͞Q-type channels (7,8). In coexpression stud...

beta-Subunits of voltage-dependent Ca(2+) channels regulate both their expression and biophysical properties. We have injected a range of concentrations of beta3-cDNA into Xenopus oocytes, with a fixed concentration of alpha1B (Ca(V)2.2) cDNA, and have quantified the corresponding linear increase of beta3 protein. The concentration dependence of a number of beta3-dependent processes has been studied. First, the dependence of the a1B maximum conductance on beta3-protein occurs with a midpoint around the endogenous concentration of beta3 (approximately 17 nM). This may represent the interaction of the beta-subunit, responsible for trafficking, with the I-II linker of the nascent channel. Second, the effect of beta3-subunits on the voltage dependence of steady-state inactivation provides evidence for two channel populations, interpreted as representing alpha1B without or with a beta3-subunit, bound with a lower affinity of 120 nM. Third, the effect of beta3 on the facilitation rate of G-protein-modulated alpha1B currents during a depolarizing prepulse to +100 mV provides evidence for the same two populations, with the rapid facilitation rate being attributed to Gbetagamma dissociation from the beta-subunit-bound alpha1B channels. The data are discussed in terms of two hypotheses, either binding of two beta-subunits to the alpha1B channel or a state-dependent alteration in affinity of the channel for the beta-subunit.