Michel De Waard

Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function.

Voltage-dependent Ca2+ channels are heteromeric complexes found in the plasma membrane of virtually all cell types and show a high level of electrophysiological and pharmacological diversity. Associated with the pore-forming alpha 1 subunit are the membrane anchored, largely extracellular alpha2-delta, the cytoplasmic beta and sometimes a transmembrane gamma subunit; these subunits dramatically influence the properties and surface expression of these channels. Effects vary depending on subunit isoforms, suggesting that functional diversity of native channels reflects heterogeneity of combinations. Interaction sites between subunits have been identified and advances have been made in our understanding of the molecular basis of functional effects of the auxiliary subunits, their capacity to be regulated by G proteins, and their interaction with related cellular systems.

The I-II Loop of the Ca2+ Channel α1 Subunit Contains an Endoplasmic Reticulum Retention Signal Antagonized by the β Subunit

Abstract The auxiliary β subunit is essential for functional expression of high voltage-activated Ca 2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of α 1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the α 1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of α 1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The β subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length α 1 subunit facilitates the cell surface expression of the channel in the absence of β subunit. Our data suggest that the β subunit favors Ca 2+ channel plasma membrane expression by inhibiting an expression brake contained in β-binding α 1 sequences.

Coding and Noncoding Variation of the Human Calcium-Channel β4-Subunit Gene CACNB4 in Patients with Idiopathic Generalized Epilepsy and Episodic Ataxia

Inactivation of the beta4 subunit of the calcium channel in the mouse neurological mutant lethargic results in a complex neurological disorder that includes absence epilepsy and ataxia. To determine the role of the calcium-channel beta4-subunit gene CACNB4 on chromosome 2q22-23 in related human disorders, we screened for mutations in small pedigrees with familial epilepsy and ataxia. The premature-termination mutation R482X was identified in a patient with juvenile myoclonic epilepsy. The R482X protein lacks the 38 C-terminal amino acids containing part of an interaction domain for the alpha1 subunit. The missense mutation C104F was identified both in a German family with generalized epilepsy and praxis-induced seizures and in a French Canadian family with episodic ataxia. These coding mutations were not detected in 255 unaffected control individuals (510 chromosomes), and they may be considered candidate disease mutations. The results of functional tests of the truncated protein R482X in Xenopus laevis oocytes demonstrated a small decrease in the fast time constant for inactivation of the cotransfected alpha1 subunit. Further studies will be required to evaluate the in vivo consequences of these mutations. We also describe eight noncoding single-nucleotide substitutions, two of which are present at polymorphic frequency, and a previously unrecognized first intron of CACNB4 that interrupts exon 1 at codon 21.

Dual Function of the Voltage-Dependent Ca2+ Channel α2δ Subunit in Current Stimulation and Subunit Interaction

Abstract Voltage-dependent Ca 2+ channels are modulated by complex interactions with the α 2 δ subunit. In vitro translation was used to demonstrate a single transmembrane topology of the α 2 δ subunit in which all but the transmembrane sequence and 5 carboxy-terminal amino acids are extracellular. The glycosylated extracellular domain is required for current stimulation, as shown by coexpression of truncated α 2 δ subunits with α 1A and β 4 subunits in Xenopus oocytes and deglycosylation with peptide-N-glycosidase F. However, coexpression of the transmembrane domain-containing δ subunit reduced the stimulatory effects of full-length α 2 δ subunits and substitution of a different transmembrane domain resulted in a loss of current stimulation. These results support a model whereby the α 2 δ transmembrane domain mediates subunit interactions and the glycosylated extracellular domain enhances current amplitude.

Ca2+ channel regulation by a conserved β subunit domain

Abstract The β subunit is a cytoplasmic component that normalizes the current amplitude, kinetics, and voltage dependence of voltage-gated Ca 2+ channels. Here, we identify a 30 amino acid domain of the β subunit that is sufficient to induce a stimulation and shift in the voltage dependence of activation of the Ca 2+ channel currents. This domain is located at the amino terminus of the second region of high conservation among all β subunit gene products. Single point mutations within this region on the β 1b subunit modified or abolished the stimulation of Ca 2+ channel currents and the binding of the β subunit to the α 1A subunit. The binding of this domain is also required for the observed changes in kinetics and voltage dependence of steady-state inactivation induced by β subunits.

Diversity of folds in animal toxins acting on ion channels

Animal toxins acting on ion channels of excitable cells are principally highly potent short peptides that are present in limited amounts in the venoms of various unrelated species, such as scorpions, snakes, sea anemones, spiders, insects, marine cone snails and worms. These toxins have been used extensively as invaluable biochemical and pharmacological tools to characterize and discriminate between the various ion channel types that differ in ionic selectivity, structure and/or cell function. Alongside the huge molecular and functional diversity of ion channels, a no less impressive structural diversity of animal toxins has been indicated by the discovery of an increasing number of polypeptide folds that are able to target these ion channels. Indeed, it appears that these peptide toxins have evolved over time on the basis of clearly distinct architectural motifs, in order to adapt to different ion channel modulating strategies (pore blockers compared with gating modifiers). Herein, we provide an up-to-date overview of the various types of fold from animal toxins that act on ion channels selective for K+, Na+, Ca2+ or Cl- ions, with special emphasis on disulphide bridge frameworks and structural motifs associated with these peptide folds.

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC β-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The β construct beta-AID dominant negative, which disrupts the RIM1-β association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with β in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

Structural and Functional Diversity of Voltage-Activated Calcium Channels

Data gathered from the expression of cDNAs that encode the subunits of voltage-dependent Ca2+ channels have demonstrated important structural and functional similarities among these channels. Despite these convergences, there are also significant differences in the nature and functional importance of subunit-subunit and protein-Ca2+ channel interactions. There is evidence demonstrating that the functional differences between Ca2+ channel subtypes is due to several factors, including the expression of distinct alpha 1 subunit proteins, the selective association of structural subunits and modulatory proteins, and differences in posttranslational processing and cell regulation. We summarize several avenues of research that should provide significant clues about the structural features involved in the biophysical and functional diversity of voltage-dependent Ca2+ channels.

A β4 Isoform-specific Interaction Site in the Carboxyl-terminal Region of the Voltage-dependent Ca2+ Channel α1A Subunit

The voltage-gated calcium channel β subunit is a cytoplasmic protein that stimulates activity of the channel-forming subunit, α1, in several ways. Complementary binding sites on α1 and β have been identified that are highly conserved among isoforms of the two subunits, but this interaction alone does not account for all of the functional effects of the β subunit. We describe here the characterization in vitro of a second interaction, involving the carboxyl-terminal cytoplasmic domain of α1A and showing specificity for the β4 (and to a lesser extent β2a) isoform. A deletion and chimera approach showed that the carboxyl-terminal region of β4, poorly conserved between β isoforms, contains the interaction site and plays a role in the regulation of channel inactivation kinetics. This is the first demonstration of a molecular basis for the specificity of functional effects seen for different combinations of these two channel components.

IDENTIFICATION OF THREE SUBUNITS OF THE HIGH AFFINITY OMEGA -CONOTOXIN MVIIC-SENSITIVE CA2+ CHANNEL

N-, P- and Q-type voltage-dependent Ca2+ channels control neurotransmitter release in the nervous system and are blocked by ω-conotoxin MVIIC. In this study, both a high affinity and a low affinity binding site for ω-conotoxin MVIIC were detected in rabbit brain. The low affinity binding site is shown to be present on the N-type Ca2+ channel. Using optimized conditions for specific labeling of the high affinity ω-conotoxin MVIIC receptor and a panel of subunit specific antibodies, the molecular structure of the high affinity receptor was investigated. We demonstrate for the first time that this receptor is composed of at least α1A, α2δ, and any one of the four brain β subunits. Such association of different β subunits with α1A and α2δ components may produce Ca2+ channels with distinct functional properties, such as P- and Q-type.