Forward genetic screen of mouse reveals dominant missense mutation in the P/Q-type voltage-dependent calcium channel, CACNA1A

Dominant mutations of the P/Q-type Ca(2+) channel (CACNA1A) underlie several human neurological disorders, including episodic ataxia type 2, familial hemiplegic migraine 1 (FHM1) and spinocerebellar ataxia 6, but have not been found previously in the mouse. Here we report the first dominant ataxic mouse model of Cacna1a mutation. This Wobbly mutant allele of Cacna1a was identified in an ethylnitrosourea (ENU) mutagenesis dominant behavioral screen. Heterozygotes exhibit ataxia from 3 weeks of age and have a normal life span. Homozygotes have a righting reflex defect from postnatal day 8 and later develop severe ataxia and die prematurely. Both heterozygotes and homozygotes exhibit cerebellar atrophy with focal reduction of the molecular layer. No obvious loss of Purkinje cells or decrease in size of the granule cell layer was observed. Real-time polymerase chain reaction revealed altered expression levels of Cacna1g, Calb2 and Th in Wobbly cerebella, but Cacna1a messenger RNA and protein levels were unchanged. Positional cloning revealed that Wobbly mice have a missense mutation leading to an arginine to leucine (R1255L) substitution, resulting in neutralization of a positively charged amino acid in repeat III of voltage sensor segment S4. The dominance of the Wobbly mutation more closely resembles patterns of CACNA1A mutation in humans than previously described mouse recessive mutants (tottering, leaner, rolling Nagoya and rocker). Positive-charge neutralization in S4 has also been shown to underlie several cases of human dominant FHM1 with ataxia. The Wobbly mutant thus highlights the importance of the voltage sensor and provides a starting point to unravel the neuropathological mechanisms of this disease.     

Conditional inactivation of the Cacna1a gene in transgenic mice

Ca(v)2.1 (P/Q-type) voltage-gated calcium channels play an important role in neurotransmitter release at many brain synapses and at the neuromuscular junction. Mutations in the CACNA1A gene, encoding the pore forming alpha(1) subunit of Ca(v)2.1 channels, are associated with a wide spectrum of neurological disorders. Here we generated mice with a conditional, floxed, Cacna1a allele without any overt phenotype. Deletion of the floxed Cacna1a allele resulted in ataxia, dystonia, and lethality during the fourth week, a severe phenotype similar to conventional Ca(v)2.1 knockout mice. Although neurotransmitter release at the neuromuscular junction was not affected in the conditional mice, homozygous deletion of the floxed allele caused an ablation of Ca(v)2.1 channel-mediated neurotransmission that was accompanied by a compensatory upregulation of Ca(v)2.3 (R-type) channels at this synapse. Pharmacological inhibition of Ca(v)2.1 channels is possible, but the contributing cell-types and time windows relevant to the different Ca(v)2.1-related neurological disorders can only be reliably determined using Cacna1a conditional mice.     

Effects of congenital stationary night blindness type 2 mutations R508Q and L1364H on Cav1.4 L-type Ca2+ channel function and expression

At least 48 mutations in the CACNA1F gene encoding retinal Ca(v)1.4 L-type Ca(2+) channels have been linked to X-linked recessive congenital stationary night blindness type 2 (CSNB2). A large number of these are missense mutations encoding full-length alpha1-subunits that can potentially form functional channels. We have previously shown that such missense mutations can confer their phenotype by different pathological mechanisms, such as complete lack of alpha1 subunit protein expression or dramatic changes in channel gating. Here we investigated the functional consequences of CSNB2 missense mutations R508Q and L1364H. We found no (R508Q) or only minor (L1364H) changes in the gating properties of both mutants after heterologous expression in Xenopus laevis oocytes (at 20 degrees C). However, both mutants resulted in altered expression density of Ca(v)1.4 currents. When expressed in the mammalian cell line tsA-201, the current amplitude of L1364H channels was reduced when cells were grown at 30 degrees C and both mutations affected total alpha1 protein expression. This effect was temperature dependent. Our data provide evidence that, in contrast to previously characterized CSNB2 missense mutations, the clinical phenotype of R508Q and L1364H is unlikely to be explained by changes in channel gating. Instead, these mutations affect the protein expression of Ca(v)1.4 Ca(2+) channels.     

Genomic structure and functional expression of a human alpha(2)/delta calcium channel subunit gene (CACNA2).

CACNA2 encodes the alpha(2)/delta subunit of the human voltage-gated calcium channels and is located in the candidate region of malignant hyperthermia susceptibility type 3 (MHS3). We determined the structural organization of CACNA2 by isolation of overlapping genomic DNA clones from a human phage library. The gene consists of at least 40 exons, 2 of which are alternatively spliced, spanning more than 150 kb of genomic DNA. Exons range from 21 to 159 bp, and introns range from 98 bp to at least more than 20 kb. We constructed a full-length cDNA and cloned it into a mammalian expression vector. Cotransfection of the CACNA2 cDNA with alpha(1A) and beta(4) cDNA into HEK293 cells led to the expression of Q-type calcium currents. The alpha(2)/delta subunit enhanced the current density 18-fold compared to cells transfected with only alpha(1A) and beta(4) cDNA. The sequence analysis provides the basis for comprehensive mutation screening of CACNA2 for putative MHS3 individuals and patients with other channelopathies.     

Mutation in the auxiliary calcium-channel subunit CACNA2D4 causes autosomal recessive cone dystrophy

Retinal signal transmission depends on the activity of high voltage-gated l-type calcium channels in photoreceptor ribbon synapses. We recently identified a truncating frameshift mutation in the Cacna2d4 gene in a spontaneous mouse mutant with profound loss of retinal signaling and an abnormal morphology of ribbon synapses in rods and cones. The Cacna2d4 gene encodes an l-type calcium-channel auxiliary subunit of the alpha (2) delta type. Mutations in its human orthologue, CACNA2D4, were not yet known to be associated with a disease. We performed mutation analyses of 34 patients who received an initial diagnosis of night blindness, and, in two affected siblings, we detected a homozygous nucleotide substitution (c.2406C-->A) in CACNA2D4. The mutation introduces a premature stop codon that truncates one-third of the corresponding open reading frame. Both patients share symptoms of slowly progressing cone dystrophy. These findings represent the first report of a mutation in the human CACNA2D4 gene and define a novel gene defect that causes autosomal recessive cone dystrophy.     

Cone dystrophy and ectopic synaptogenesis in a Cacna1f loss of function model of congenital stationary night blindness (CSNB2A)

Congenital stationary night blindness 2A (CSNB2A) is an X-linked retinal disorder, characterized by phenotypically variable signs and symptoms of impaired vision. CSNB2A is due to mutations in CACNA1F, which codes for the pore-forming α_1F subunit of a L-type voltage-gated calcium channel, Ca_v1.4. Mouse models of CSNB2A, used for characterizing the effects of various Cacna1f mutations, have revealed greater severity of defects than in human CSNB2A. Specifically, Cacna1f-knockout mice show an apparent lack of visual function, gradual retinal degeneration, and disruption of photoreceptor synaptic terminals. Several reports have also noted cone-specific disruptions, including axonal abnormalities, dystrophy, and cell death. We have explored further the involvement of cones in our ‘G305X’ mouse model of CSNB2A, which has a premature truncation, loss-of-function mutation in Cacna1f. We show that the expression of genes for several phototransduction-related cone markers is down-regulated, while that of several cellular stress- and damage-related markers is up-regulated; and that cone photoreceptor structure and photopic visual function – measured by immunohistochemistry, optokinetic response and electroretinography – deteriorate progressively with age. We also find that dystrophic cone axons establish synapse-like contacts with rod bipolar cell dendrites, which they normally do not contact in wild-type retinas – ectopically, among rod cell bodies in the outer nuclear layer. These data support a role for Ca_v1.4 in cone synaptic development, cell viability, and synaptic transmission of cone-dependent visual signals. Although our novel finding of cone-to-rod-bipolar cell contacts in this mouse model of a retinal channelopathy may challenge current views of the role of Ca_v1.4 in photopic vision, it also suggests a potential new target for restorative therapy.     

Functional properties of a new voltage-dependent calcium channel alpha(2)delta auxiliary subunit gene (CACNA2D2)

We have positionally cloned and characterized a new calcium channel auxiliary subunit, alpha(2)delta-2 (CACNA2D2), which shares 56% amino acid identity with the known alpha(2)delta-1 subunit. The gene maps to the critical human tumor suppressor gene region in chromosome 3p21.3, showing very frequent allele loss and occasional homozygous deletions in lung, breast, and other cancers. The tissue distribution of alpha(2)delta-2 expression is different from alpha(2)delta-1, and alpha(2)delta-2 mRNA is most abundantly expressed in lung and testis and well expressed in brain, heart, and pancreas. In contrast, alpha(2)delta-1 is expressed predominantly in brain, heart, and skeletal muscle. When co-expressed (via cRNA injections) with alpha(1B) and beta(3) subunits in Xenopus oocytes, alpha(2)delta-2 increased peak size of the N-type Ca(2+) currents 9-fold, and when co-expressed with alpha(1C) or alpha(1G) subunits in Xenopus oocytes increased peak size of L-type channels 2-fold and T-type channels 1.8-fold, respectively. Anti-peptide antibodies detect the expression of a 129-kDa alpha(2)delta-2 polypeptide in some but not all lung tumor cells. We conclude that the alpha(2)delta-2 gene encodes a functional auxiliary subunit of voltage-gated Ca(2+) channels. Because of its chromosomal location and expression patterns, CACNA2D2 needs to be explored as a potential tumor suppressor gene linking Ca(2+) signaling and lung, breast, and other cancer pathogenesis. The homologous location on mouse chromosome 9 is also the site of the mouse neurologic mutant ducky (du), and thus, CACNA2D2 is also a candidate gene for this inherited idiopathic generalized epilepsy syndrome.     

Photoreceptor Degeneration in Two Mouse Models for Congenital Stationary Night Blindness Type 2

Light-dependent conductance changes of voltage-gated Ca_v1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Ca_v1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Ca_v1.4 channels, but some mutations alter the channels’ gating properties and, presumably, disturb Ca²⁺ influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14–17) in a longitudinal study up to eight months of age. In ΔEx14–17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca²⁺ responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca²⁺ response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14–17 mutants.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Expression of purinergic P2X2 receptor-channels and their role in calcium signaling in pituitary cells.

Pituitary cells express purinergic receptor-channels (P2XR), the activation of which by ATP is associated with the facilitation of Ca2+ influx. Pharmacological, RT-PCR, and nucleotide sequence analyses confirm the presence of a wild type P2X2aR and a spliced isoform P2X2bR, which lacks a portion of carboxyl terminal amino acids. Wild type and spliced isoform receptors have a similar EC50 for ATP and time-course for activation, but the spliced isoform exhibits rapid and complete desensitization, whereas the wild type channel desensitizes slowly and incompletely. Deletion and insertion studies have revealed that a 6 residue sequence located in carboxyl tail (Arg371-Pro376) is required for sustained Ca2+ influx through wild type receptors. When co-expressed, the wild type and spliced channels form functional heteropolymeric channels. The patterns of Ca2+ signaling in the majority of pituitary cells expressing ATP-gated receptor-channels are highly comparable to those observed in cells co-transfected with P2X2aR and P2X2bR. ATP-induced [Ca2+]i response in pituitary cells is partially inhibited by nifedipine, a blocker of voltage-gated L-type Ca2+ channels, suggesting that P2X2R not only drive Ca2+ into the cell, but also activate voltage-gated Ca2+ entry. Our results indicate that ATP represents a paracrine and (or) autocrine factor in the regulation of Ca2+ signaling, and that its actions are mediated in part by heteropolymeric P2X2R.