Altered expression of the voltage-gated calcium channel subunit α2δ-1: a comparison between two experimental models of epilepsy and a sensory nerve ligation model of neuropathic pain.

The auxiliary α2δ-1 subunit of voltage-gated calcium channels is up-regulated in dorsal root ganglion neurons following peripheral somatosensory nerve damage, in several animal models of neuropathic pain. The α2δ-1 protein has a mainly presynaptic localization, where it is associated with the calcium channels involved in neurotransmitter release. Relevant to the present study, α2δ-1 has been shown to be the therapeutic target of the gabapentinoid drugs in their alleviation of neuropathic pain. These drugs are also used in the treatment of certain epilepsies. In this study we therefore examined whether the level or distribution of α2δ-1 was altered in the hippocampus following experimental induction of epileptic seizures in rats, using both the kainic acid model of human temporal lobe epilepsy, in which status epilepticus is induced, and the tetanus toxin model in which status epilepticus is not involved. The main finding of this study is that we did not identify somatic overexpression of α2δ-1 in hippocampal neurons in either of the epilepsy models, unlike the upregulation of α2δ-1 that occurs following peripheral nerve damage to both somatosensory and motor neurons. However, we did observe local reorganisation of α2δ-1 immunostaining in the hippocampus only in the kainic acid model, where it was associated with areas of neuronal cell loss, as indicated by absence of NeuN immunostaining, dendritic loss, as identified by areas where microtubule-associated protein-2 immunostaining was missing, and reactive gliosis, determined by regions of strong OX42 staining

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. Our main findings are that there is reduced Ca(2+) entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α2δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca(2+) buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α2δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca(2+)-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca(2+) elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α2δ-1 knockout sensory neurons.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'

A Ca(V)2.1 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. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide

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 α₂δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α₂δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2du2J mutation results in a 2 bp deletion in the coding region and a complete loss of α₂δ-2 protein. Here we show that du^{2J}/du^{2J} mice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C, du^{2J}/du^{2J} PCs show no spontaneous intrinsic activity. DU^{2J}/du^{2J} mice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du^{2J}/+ mice have a marked reduction in α₂δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However, du^{2J}/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α₂δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α₂δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma

N terminus is key to the dominant negative suppression of CaV2 calcium channels: implications for episodic ataxia type 2

Expression of the calcium channels CaV2.1 and CaV2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in CaV2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both CaV2.1 and CaV2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg52 and Arg54) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of CaV2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in CaV2.2 protein level, and this is also prevented by mutation of Arg52 and Arg54 to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg52, Arg54 motif are involved in the processes underlying dominant negative suppression

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 (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS

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

AbstractEpisodic 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. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide

Ablation of α_{2}δ-1 inhibits cell-surface trafficking of endogenous N-type calcium channels in the pain pathway in vivo

The auxiliary α_{2}δ calcium channel subunits play key roles in voltage-gated calcium channel function. Independent of this, α_{2}δ-1 has also been suggested to be important for synaptogenesis. Using an epitope-tagged knockin mouse strategy, we examined the effect of α_{2}δ-1 on Ca_{V}2.2 localization in the pain pathway in vivo, where Ca_{V}2.2 is important for nociceptive transmission and α_{2}δ-1 plays a critical role in neuropathic pain. We find Ca_{V}2.2 is preferentially expressed on the plasma membrane of calcitonin gene-related peptide-positive small nociceptors. This is paralleled by strong presynaptic expression of Ca_{V}2.2 in the superficial spinal cord dorsal horn. EM-immunogold localization shows Ca_{V}2.2 predominantly in active zones of glomerular primary afferent terminals. Genetic ablation of α_{2}δ-1 abolishes Ca_{V}2.2 cell-surface expression in dorsal root ganglion neurons and dramatically reduces dorsal horn expression. There was no effect of α2δ-1 knockout on other dorsal horn pre- and postsynaptic markers, indicating the primary afferent pathways are not otherwise affected by α_{2}δ-1 ablation

Proteolytic maturation of α 2 δ represents a checkpoint for activation and neuronal trafficking of latent calcium channels

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes

Proteolytic maturation of alpha(2)delta represents a checkpoint for activation and neuronal trafficking of latent calcium channels

The auxiliary a2d subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides a2 and d. We now show, using a2d constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved a2d inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of a2d, voltage-dependent activation of channels is promoted, independent from the trafficking role of a2d. Uncleaved a2d does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved a2d subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of a2d then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes