7 3 a r t I C l e SThe level of expression of voltage-gated calcium channels at the plasma membrane is a key regulator of calcium homeostasis in excitable cells, and of downstream effects such as calcium-dependent transcription 1,2 . Members of the high voltage-activated (HVA) calcium channel family are heteromultimeric protein complexes that contain a pore-forming α 1 subunit that defines the channel subtype, plus ancillary α 2 -δ and β subunits that not only alter the function of the α 1 subunit but also regulate the trafficking of the channel complex to the plasma membrane 3-8 . The mammalian genome encodes four different types of Cavβ subunit that have distinct spatial and temporal expression patterns [4][5][6] . With the exception of Cavβ 2a , these subunits are cytoplasmic proteins that physically bind to a region in the α 1 subunit domain I-II linker that is highly conserved among all HVA calcium channels and is termed the alpha interaction domain (AID) 7 . Crystal structure data show that the Cavβ subunit contains interacting SH3 and guanylate kinase domains, with the latter participating in high-affinity binding to the AID region [8][9][10] . The physiological consequences of this interaction are underscored by gene knockout studies showing that deletion of the Cavβ 1a or Cavβ 2a subunits causes embryonic lethality 11,12 and by findings that a premature stop mutation in Cavβ 4 causes an epileptic phenotype in mice 13 .It has been suggested that the Cavβ subunit masks an endoplasmic reticulum retention signal on the Cav2.1 α 1 subunit 14 , thereby leading to increased cell surface expression of P/Q-type channels. However, no specific endoplasmic reticulum retention motif in the AID and surrounding regions of the α 1 subunit has been identified, and removing the AID motif in the I-II linker of Cav2.1 does not increase current amplitude in the absence of Cavβ (ref. 15). Moreover, it is not clear whether different HVA calcium channel isoforms share common retention motifs. Here we show that Cav1.2 (L-type) calcium channels contain an endoplasmic reticulum retention motif in the proximal C-terminal region, and we provide evidence that the Cavβ subunit has a role in regulating proteasomal degradation of these channels. Our data show that the Cavβ subunit acts as a molecular switch that prevents the ubiquitination of the channels and their targeting to the ERAD complex and thereby regulates their expression at the plasma membrane. RESULTS Cavb regulates endoplasmic reticulum retention of Cav1.2We first performed an ELISA assay involving a Cav1.2 channel construct tagged with an extracellular hemagglutinin (HA) epitope (Fig. 1a). We compared immunoluminescence between permeabilized and nonpermeabilized cells, which allowed us to quantify the relative proportion of Cav1.2 channels that were inserted into the plasma membrane. Coexpression with the Cavβ 1b or Cavβ 2a subunit mediated a significant increase in the fraction of Cav1.2 channels at the cell surface (Fig. 1a and data not shown). This was confirmed by HA...

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We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker -atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro 10 , Asn 27 , and Arg 35 , that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled -atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 M, -atracotoxinHv1a had no effect on heterologously expressed rat Ca v 2.1, Ca v 2.2, and Ca v 1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 M -atracotoxin-Hv1a inhibited rat Ca v currents by 10 -34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn 27 and Arg 35 , which appears to be critical for interaction of the toxin with insect Ca v channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro 10 , Asn 27 , and Arg 35 confers specific binding to insect Ca v channels and is largely responsible for the remarkable phyletic selectivity of -atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Ca v channels.The first peptide neurotoxins isolated from the venom of Australian funnel-web spiders (genera Atrax and Hadronyche) and shown to have selective activity against insects were members of the -atracotoxin-1 (ACTX) 1 family (1-3). These toxins comprise 36 -37 residues with six strictly conserved cysteine residues that are paired to form three disulfide bridges (4). The best studied family member, -ACTX-Hv1a, is one of the most potent insecticidal peptide toxins discovered so far (4, 5); it has proved lethal to all insect orders that have been tested, including coleopterans, dictyopterans, hemipterans, orthopterans, and refractory lepidopteran pests such as the tobacco budworm Heliothis virescens and the cotton bollworm Helicoverpa armigera (1, 2, 5).The phyletic specificity of these toxins appears to reside in their ability to block insect, but not vertebrate, voltage-gated calcium channels (2). Although nanomolar concentrations of -ACTX-Hv1a are sufficient to block high voltage-activated (HVA) calcium currents in the central nervous system of the fruit fly Drosophila melanogaster and the American cockroach Periplaneta americana (2, 5), 1 M toxin does not block HVA calcium currents in vertebrate neurons (2) and the toxin is harmless when injected subcutaneously into newborn mice (1).With the exception of the organophosphate and carbamate insecticides (which target acetylcholinesterase), the vast majority of synthetic insecticides are directed against voltagegated sodium channels, nicotinic acetylcholine receptors, or ...