The Interplay Between Splicing of Two Exon Combinations Differentially Affects Membrane Targeting and Function of Human Ca2.2.

N-type calcium channels (Ca2.2) are predominantly localized in presynaptic terminals, and are particularly important for pain transmission in the spinal cord. Furthermore, they have multiple isoforms, conferred by alternatively spliced or cassette exons, which are differentially expressed.

Nerve injury increases native Ca V 2.2 trafficking in dorsal root ganglion mechanoreceptors.

Neuronal N-type (Ca V 2.2) voltage-gated calcium channels are essential for neurotransmission from primary afferent terminals in the dorsal horn. In this study, we have used a knockin mouse containing Ca V 2.

Ablation of αδ-1 inhibits cell-surface trafficking of endogenous N-type calcium channels in the pain pathway in vivo.

The auxiliary αδ calcium channel subunits play key roles in voltage-gated calcium channel function. Independent of this, αδ-1 has also been suggested to be important for synaptogenesis. Using an epitope-tagged knockin mouse strategy, we examined the effect of αδ-1 on Ca2.

LRP1 influences trafficking of N-type calcium channels via interaction with the auxiliary αδ-1 subunit.

Voltage-gated Ca (Ca) channels consist of a pore-forming α1 subunit, which determines the main functional and pharmacological attributes of the channel. The Ca1 and Ca2 channels are associated with auxiliary β- and αδ-subunits. The molecular mechanisms involved in αδ subunit trafficking, and the effect of αδ subunits on trafficking calcium channel complexes remain poorly understood.

Proteolytic maturation of αδ represents a checkpoint for activation and neuronal trafficking of latent calcium channels.

The auxiliary αδ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α and δ. We now show, using αδ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (Ca2.2) calcium channels.

Effect of knockout of α2δ-1 on action potentials in mouse sensory neurons.

Gene deletion of the voltage-gated calcium channel auxiliary subunit α2δ-1 has been shown previously to have a cardiovascular phenotype, and a reduction in mechano- and cold sensitivity, coupled with delayed development of neuropathic allodynia. We have also previously shown that dorsal root ganglion (DRG) neuron calcium channel currents were significantly reduced in α2δ-1 knockout mice. To extend our findings in these sensory neurons, we have examined here the properties of action potentials (APs) in DRG neurons from α2δ-1 knockout mice in comparison to their wild-type (WT) littermates, in order to dissect how the calcium channels that are affected by α2δ-1 knockout are involved in setting the duration of individual APs and their firing frequency.

A CaV2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant.

Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins.

Thrombospondin-4 reduces binding affinity of [(3)H]-gabapentin to calcium-channel α2δ-1-subunit but does not interact with α2δ-1 on the cell-surface when co-expressed.

The α2δ proteins are auxiliary subunits of voltage-gated calcium channels, and influence their trafficking and biophysical properties. The α2δ ligand gabapentin interacts with α2δ-1, and inhibits calcium channel trafficking. However, α2-1 has also been proposed to play a synaptogenic role, independent of calcium channel function.

Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density.

Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (Ca(V)) channels. Here we show that the functional expression of neuronal N-type Ca(V) channels (Ca(V)2.

α2δ-1 gene deletion affects somatosensory neuron function and delays mechanical hypersensitivity in response to peripheral nerve damage.

The α2δ-1 subunit of voltage-gated calcium channels is upregulated after sensory nerve injury and is also the therapeutic target of gabapentinoid drugs. It is therefore likely to play a key role in the development of neuropathic pain. In this study, we have examined mice in which α2δ-1 gene expression is disrupted, to determine whether α2δ-1 is involved in various modalities of nociception, and for the development of behavioral hypersensitivity after partial sciatic nerve ligation (PSNL).

The alpha2delta subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function.

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the alpha1 subunit is associated with two auxiliary subunits, the intracellular beta subunit and the alpha(2)delta subunit; both of these subunits influence the trafficking and properties of Ca(V)1 and Ca(V)2 channels. The alpha(2)delta subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the alpha(2)delta proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to delta rather than a transmembrane domain.

N terminus is key to the dominant negative suppression of Ca(V)2 calcium channels: implications for episodic ataxia type 2.

Expression of the calcium channels Ca(V)2.1 and Ca(V)2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I.

Labelling of the 3D structure of the cardiac L-type voltage-gated calcium channel.

Voltage-gated calcium channels (VGCCs) regulate calcium influx into all excitable cells. In the heart, the main calcium channels are the L-type VGCCs (LTCCs). These are localised to the sarcolemmal membrane, and are hetero-oligomeric complexes comprised of three non-covalently associated polypeptides; alpha1 (CaV1.

The increased trafficking of the calcium channel subunit alpha2delta-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin.

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit alpha(2)delta-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both alpha(2)delta ligands, have antiallodynic effects, but their mechanism of action has remained elusive.

Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin.

The mechanism of action of the antiepileptic and antinociceptive drugs of the gabapentinoid family has remained poorly understood. Gabapentin (GBP) binds to an exofacial epitope of the alpha(2)delta-1 and alpha(2)delta-2 auxiliary subunits of voltage-gated calcium channels, but acute inhibition of calcium currents by GBP is either very minor or absent. We formulated the hypothesis that GBP impairs the ability of alpha(2)delta subunits to enhance voltage-gated Ca(2+)channel plasma membrane density by means of an effect on trafficking.

The ducky(2J) mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression.

The mouse mutant ducky and its allele ducky(2J) represent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the alpha2delta-2 calcium channel subunit. Of relevance to the ataxic phenotype, alpha2delta-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs).

Interaction via a key tryptophan in the I-II linker of N-type calcium channels is required for beta1 but not for palmitoylated beta2, implicating an additional binding site in the regulation of channel voltage-dependent properties.

The CaVbeta subunits of voltage-gated calcium channels regulate these channels in several ways. Here we investigate the role of these auxiliary subunits in the expression of functional N-type channels at the plasma membrane and in the modulation by G-protein-coupled receptors of this neuronal channel. To do so, we mutated tryptophan 391 to an alanine within the alpha-interacting domain (AID) in the I-II linker of CaV2.

COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis.

Jasmonates (JAs) regulate Arabidopsis thaliana wound and defence responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. Other F-box proteins interact with SKP1 and cullin proteins to form SCF complexes that selectively recruit regulatory proteins targeted for ubiquitination.