Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.

Neonatal brain injury is a problem of global importance. To date, no causal therapies are available. A substance with considerable therapeutic potential is the endogenous neuropeptide secretoneurin (SN), which has proven to be beneficial in adult stroke. The aim of this study was to assess its effect in neonatal hypoxic-ischemic brain injury models. In vitro, primary hippocampal neurons were pre-treated with vehicle, 1µg/ml, 10µg/ml, or 50µg/ml SN and subjected to oxygen-glucose deprivation (OGD) for six hours. Cell death was assessed after a 24-h recovery period. In vivo, seven day-old CD-1 mice underwent unilateral common carotid artery ligation and were exposed to 8% oxygen/nitrogen for 20 min. SN plasma concentrations were serially determined by ELISA after insult. One hour after hypoxia, a subgroup of animals was treated with vehicle or SN. SN plasma concentrations significantly decreased 48h after insult. The number of caspase-3-positive cells was significantly lower in the hypoxic-ischemic hemisphere in the thalamus of SN-treated animals. In the hypoxic-only hemisphere administration of SN significantly reduced the number of caspase-3-positive cells (in cortex, white matter, hippocampus, thalamus and striatum) and inhibited microglial cell activation in the thalamus. SN has neuroprotective potential in neonatal brain injury. Its main action seems to be inhibition of apoptosis in the aftermath of the insult, predominantly in the hypoxic-only hemisphere. This might be explained by the less pronounced injury in this hemisphere, where blood flow and thus nutrient supply are maintained.

Genetic polymorphisms of the L-type voltage-gated calcium channel (VGCC) are associated with psychiatric disorders including major depressive disorder. Alterations of S100A10 (p11) level are also implicated in the etiology of major depressive disorder. However, the existence of an endogenous regulator in the brain regulating p11, L-type VGCC, and depressive behavior has not been known. Here we report that Ahnak, whose function in the brain has been obscure, stabilizes p11 and Anxa2 proteins in the hippocampus and prefrontal cortex in the rodent brain. Protein levels of Ahnak, p11, and Anxa2 are highly and positively correlated in the brain. Together these data suggest the existence of an Ahnak/p11/Anxa2 protein complex. Ahnak is expressed in p11-positive as well as p11-negative neurons. Ahnak, through its N-terminal region, scaffolds the L-type pore-forming α1 subunit and, through its C-terminal region, scaffolds the β subunit of VGCC and the p11/Anxa2 complex. Cell surface expression of the α1 subunits and L-type calcium current are significantly reduced in primary cultures of Ahnak knockout (KO) neurons compared to wild-type controls. A decrease in the L-type calcium influx is observed in both glutamatergic neurons and parvalbumin (PV) GABAergic interneurons of Ahnak KO mice. Constitutive Ahnak KO mice or forebrain glutamatergic neuron-selective Ahnak KO mice display a depression-like behavioral phenotype similar to that of constitutive p11 KO mice. In contrast, PV interneuron-selective Ahnak KO mice display an antidepressant-like behavioral phenotype. Our results demonstrate L-type VGCC as an effector of the Ahnak/p11/Anxa2 complex, revealing a novel molecular connection involved in the control of depressive behavior.

Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the CACNA1A gene. As one of the few bicistronic genes discovered in the human genome, CACNA1A encodes not only the α1A subunit of the P/Q type voltage-gated Ca2+ channel CaV2.1 but also the α1ACT protein, a 75 kDa transcription factor sharing the sequence of the cytoplasmic C-terminal tail of the α1A subunit. Isoforms of both proteins contain the polyglutamine (polyQ) domain that is expanded in SCA6 patients. Although certain SCA6 phenotypes appear to be specific for Purkinje neurons, other pathogenic effects of the SCA6 polyQ mutation can affect a broad spectrum of central nervous system (CNS) neuronal subtypes. We investigated the expression and function of CACNA1A gene products in human neurons derived from induced pluripotent stem cells from two SCA6 patients. Expression levels of CACNA1A encoding α1A subunit were similar between SCA6 and control neurons, and no differences were found in the subcellular distribution of CaV2.1 channel protein. The α1ACT immunoreactivity was detected in the majority of cell nuclei of SCA6 and control neurons. Although no SCA6 genotype-dependent differences in CaV2.1 channel function were observed, they were found in the expression levels of the α1ACT target gene Granulin (GRN) and in glutamate-induced cell vulnerability.

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and β4 subunits. β4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β4b cDNA. Heterologously expressed wild-type β4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, β4b and β4b-L125P augmented the calcium current amplitudes, however, β4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β4b in cultured myotubes and hippocampal neurons. While binding of β4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of β4b.

Auxiliary α2δ subunits of voltage-gated calcium channels modulate channel trafficking, current properties, and synapse formation. Three of the four isoforms (α2δ-1, α2δ-2, and α2δ-3) are abundantly expressed in the brain; however, of the available knockout models, only α2δ-2 knockout or mutant mice display an obvious abnormal neurological phenotype. Thus, we hypothesize that the neuronal α2δ isoforms may have partially specific as well as redundant functions. To address this, we generated three distinct α2δ double knockout mouse models by crossbreeding single knockout (α2δ-1 and -3) or mutant (α2δ-2/ducky) mice. Here, we provide a first phenotypic description and brain structure analysis. We found that genotypic distribution of neonatal litters in distinct α2δ-1/-2, α2δ-1/-3, and α2δ-2/-3 breeding combinations did not conform to Mendel's law, suggesting premature lethality of single and double knockout mice. Notably, high occurrences of infant mortality correlated with the absence of specific α2δ isoforms (α2Δ-2 > α2δ-1 > α2δ-3), and was particularly observed in cages with behaviorally abnormal parenting animals of α2δ-2/-3 cross-breedings. Juvenile α2δ-1/-2 and α2δ-2/-3 double knockout mice displayed a waddling gate similar to ducky mice. However, in contrast to ducky and α2δ-1/-3 double knockout animals, α2δ-1/-2 and α2δ-2/-3 double knockout mice showed a more severe disease progression and highly impaired development. The observed phenotypes within the individual mouse lines may be linked to differences in the volume of specific brain regions. Reduced cortical volume in ducky mice, for example, was associated with a progressively decreased space between neurons, suggesting a reduction of total synaptic connections. Taken together, our findings show that α2δ subunits differentially regulate premature survival, postnatal growth, brain development, and behavior, suggesting specific neuronal functions in health and disease.

Retinitis Pigmentosa is a genetically heterogeneous, degenerative retinal disorder characterized by gradual dysfunction and death of photoreceptors, first rods and later cones, and progressive blindness. Studies suggested that application of L-type calcium channel blockers rescues photoreceptors in paradigms related to Ca2+ overflow. To investigate whether Cav1.3 L-type channels have protective effects in the retina, we established a new mouse model by crossing rd10, modeling autosomal-recessive RP, with Cav1.3 deficient mice (rd10/Cav1.3KO). Our immunohistochemical analyses revealed an influence of Cav1.3 channels on the degenerative process of photoreceptors. The absence of Cav1.3 delayed the centre-to-periphery degeneration of rods indicated by a significantly higher number of photoreceptor rows and, consequently, of cones. In accordance with a preserved number of cones we observed a regular row of cone somas in rd10/Cav1.3-KO retinas. Surviving rod photoreceptors maintained synaptic contacts with rod bipolar cells. However, the delay in degeneration was only observed up to postnatal day 45. Although we observed a reduction in the spontaneous oscillatory retinal activity during multielectrode array analyses, measurable functional preservation was lacking in behavioural tests. In conclusion, Cav1.3 channels contribute to photoreceptor degeneration in rd10 retinas but photoreceptor temporary rescue might rather be achieved indirectly through other retinal cell layers.

Cav1.4 L-type calcium channels are predominantly expressed at the photoreceptor terminals and in bipolar cells, mediating neurotransmitter release. Mutations in its gene, CACNA1F, can cause congenital stationary night-blindness type 2 (CSNB2). Due to phenotypic variability in CSNB2, characterization of pathological variants is necessary to better determine pathological mechanism at the site of action. A set of known mutations affects conserved gating charges in the S4 voltage sensor, two of which have been found in male CSNB2 patients. Here, we describe two disease-causing Cav1.4 mutations with gating charge neutralization, exchanging an arginine 964 with glycine (RG) or arginine 1288 with leucine (RL). In both, charge neutralization was associated with a reduction channel expression also reflected in smaller ON gating currents. In RL channels, the strong decrease in whole-cell current densities might additionally be explained by a reduction of single-channel currents. We further identified alterations in their biophysical properties, such as a hyperpolarizing shift of the activation threshold and an increase in slope factor of activation and inactivation. Molecular dynamic simulations in RL substituted channels indicated water wires in both, resting and active, channel states, suggesting the development of omega (ω)currents as a new pathological mechanism in CSNB2. This sum of the respective channel property alterations might add to the differential symptoms in patients beside other factors, such as genomic and environmental deviations.

It is well established that L-type calcium channels (LTCCs) are expressed in astroglia. However, their functional role is still speculative, especially under pathologic conditions. We recently showed that the α1 subunit-like immunoreactivity of the CaV1.2 channel is strongly expressed in reactive astrocytes around beta-amyloid plaques in 11-month-old Alzheimer transgenic (tg) mice with the amyloid precursor protein London and Swedish mutations. The aim of the present study was to examine the cellular expression of all LTCC subunits around beta-amyloid plaques by in situ hybridization using (35)S-labeled oligonucleotides. Our data show that messenger RNAs (mRNAs) of the LTCC CaV1.2 α1 subunit as well as all auxiliary β and α2δ subunits, except α2δ-4, were expressed in the hippocampus of age-matched wild-type mice. It was unexpected to see, that cells directly located in the plaque core in the cortex expressed mRNAs for CaV1.2 α1, β2, β4, and α2δ-1, whereas no expression was detected in the halo. Furthermore, cells in the plaque core also expressed preprotachykinin-A mRNA, the precursor for substance P. By means of confocal microscopy, we demonstrated that collagen-IV-stained brain vessels in the cortex were associated with the plaque core and were immunoreactive for substance P. In cortical organotypic brain slices of adult Alzheimer mice, we could demonstrate that LTCC blockers increased angiogenesis, which was further potentiated by substance P. In conclusion, our data show that brain vessels associated with beta-amyloid plaques express substance P and an LTCC and may play a role in angiogenesis.

Auxiliary beta subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca(2+) channels in heterologous cells. In brain, all four beta isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of beta subunits and their role in membrane expression of Ca(V)1.2 alpha(1) subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous beta subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged beta subunits demonstrated that all beta subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca(V)1.2, thus indicating a great promiscuity in alpha(1)-beta interactions. In contrast, restricted axonal targeting of beta(1) and weak colocalization of beta(4b) with Ca(V)1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Ca(V)1.2 was strongly enhanced by all beta subunits in an isoform-specific manner. Conversely, mutating the alpha-interaction domain of Ca(V)1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a beta subunit with the alpha-interaction domain is absolutely essential for membrane expression of alpha(1) subunits, as well as for the subcellular localization of beta subunits, which by themselves possess little or no targeting properties.

The β subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain β subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β4 subunit lacking the variable N terminus (β4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length β4 variants (β4a and β4b), β4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of β4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (β4-null) mice individually reconstituted with β4a, β4b, and β4e. Notably, the number of genes regulated by each β4 splice variant correlated with the rank order of their nuclear-targeting properties (β4b > β4a > β4e). Together, these findings support isoform-specific functions of β4 splice variants in neurons, with β4b playing a dual role in channel modulation and gene regulation, whereas the newly detected β4e variant serves exclusively in calcium-channel-dependent functions.

Voltage-gated calcium channels regulate gene expression by controlling calcium entry through the plasma membrane and by direct interactions of channel fragments and auxiliary β subunits with promoters and the epigenetic machinery in the nucleus. Mutations of the calcium channel β(4) subunit gene (CACNB4) cause juvenile myoclonic epilepsy in humans and ataxia and epileptic seizures in mice. Recently a model has been proposed according to which failed nuclear translocation of the truncated β(4) subunit R482X mutation resulted in altered transcriptional regulation and consequently in neurological disease. Here we examined the nuclear targeting properties of the truncated β(4b(1–481)) subunit in tsA-201 cells, skeletal myotubes, and in hippocampal neurons. Contrary to expectation, nuclear targeting of β(4b(1–481)) was not reduced compared with full-length β(4b) in any one of the three cell systems. These findings oppose an essential role of the β(4) distal C-terminus in nuclear targeting and challenge the idea that the nuclear function of calcium channel β(4) subunits is critically involved in the etiology of epilepsy and ataxia in patients and mouse models with mutations in the CACNB4 gene.

Presynaptic α2δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α2δ-2 can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ-2 is essential for the trans-synaptic mechanism. The novel function of α2δ-2 identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α2δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α2δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α2δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.

Cav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel's activation threshold toward more hyperpolarized potentials. Taken together, the association with "slow" β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

Voltage-gated Cav1.2 and Cav1.3 (L-type) Ca2+ channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors-phenotypes that more closely match those in mice lacking Cav1.2 than Cav1.3. Therefore, we investigated the functional impact of densin on Cav1.2. We report that densin is an essential regulator of Cav1.2 in neurons, but has distinct modulatory effects compared with its regulation of Cav1.3. Densin binds to the N-terminal domain of Cav1.2, but not that of Cav1.3, and increases Cav1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Cav1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Cav1.2 channels, overexpression of densin increases the clustering of Cav1.2 in dendrites of hippocampal neurons in culture. Compared with wild-type mice, the cell surface levels of Cav1.2 in the brain, as well as Cav1.2 current density and signaling to the nucleus, are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Cav1 channels and ensures efficient Cav1.2 Ca2+ signaling at excitatory synapses.SIGNIFICANCE STATEMENT The number and localization of voltage-gated Cav Ca2+ channels are crucial determinants of neuronal excitability and synaptic transmission. We report that the protein densin-180 is highly enriched at excitatory synapses in the brain and enhances the cell surface trafficking and postsynaptic localization of Cav1.2 L-type Ca2+ channels in neurons. This interaction promotes coupling of Cav1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that may contribute to the roles of Cav1.2 in regulating cognition and mood.

CaV1.4 L-type calcium channels are predominantly expressed in photoreceptor terminals playing a crucial role for synaptic transmission and, consequently, for vision. Human mutations in the encoding gene are associated with congenital stationary night blindness type-2. Besides rod-driven scotopic vision also cone-driven photopic responses are severely affected in patients. The present study therefore examined functional and morphological changes in cones and cone-related pathways in mice carrying the CaV1.4 gain-of function mutation I756T (CaV1.4-IT) using multielectrode array, patch-clamp and immunohistochemical analyses. CaV1.4-IT ganglion cell responses to photopic stimuli were seen only in a small fraction of cells indicative of a major impairment in the cone pathway. Though cone photoreceptors underwent morphological rearrangements, they retained their ability to release glutamate. Our functional data suggested a postsynaptic cone bipolar cell defect, supported by the fact that the majority of cone bipolar cells showed sprouting, while horizontal cells maintained contacts with cones and cone-to-horizontal cell input was preserved. Furthermore a reduction of basal Ca2+ influx by a calcium channel blocker was not sufficient to rescue synaptic transmission deficits caused by the CaV1.4-IT mutation. Long term treatments with low-dose Ca2+ channel blockers might however be beneficial reducing Ca2+ toxicity without major effects on ganglion cells responses.

-->Low voltage-activated Cav1.3 L-type Ca2+-channels are key regulators of neuronal excitability controlling neuronal development and different types of learning and memory. Their physiological functions are enabled by their negative activation voltage-range, which allows Cav1.3 to be active at subthreshold voltages. Alternative splicing in the C-terminus of their pore-forming α1-subunits gives rise to C-terminal long (Cav1.3L) and short (Cav1.3S) splice variants allowing Cav1.3S to activate at even more negative voltages than Cav1.3L. We discovered that inclusion of exons 8b, 11, and 32 in Cav1.3S further shifts activation (-3 to -4 mV) and inactivation (-4 to -6 mV) to more negative voltages as revealed by functional characterization in tsA-201 cells. We found transcripts of these exons in mouse chromaffin cells, the cochlea, and the brain. Our data further suggest that Cav1.3-containing exons 11 and 32 constitute a significant part of native channels in the brain. We therefore investigated the effect of these splice variants on human disease variants. Splicing did not prevent the gating defects of the previously reported human pathogenic variant S652L, which further shifted the voltage-dependence of activation of exon 11-containing channels by more than -12 mV. In contrast, we found no evidence for gating changes of the CACNA1D missense variant R498L, located in exon 11, which has recently been identified in a patient with an epileptic syndrome. Our data demonstrate that alternative splicing outside the C-terminus involving exons 11 and 32 contributes to channel fine-tuning by stabilizing negative activation and inactivation gating properties of wild-type and mutant Cav1.3 channels.

Auxiliary beta subunits are critical determinants of membrane expression and gating properties of voltage-gated calcium channels. Mutations in the beta(4) subunit gene cause ataxia and epilepsy. However, the specific function of beta(4) in neurons and its causal relation to neurological diseases are unknown. Here we report the localization of the beta(4) subunit in the nuclei of cerebellar granule and Purkinje cells. beta(4b) was the only beta isoform showing nuclear targeting when expressed in neurons and skeletal myotubes. Its specific nuclear targeting property was mapped to an N-terminal double-arginine motif, which was necessary and sufficient for targeting beta subunits into the nucleus. Spontaneous electrical activity and calcium influx negatively regulated beta(4b) nuclear localization by a CRM-1-dependent nuclear export mechanism. The activity-dependent shuttling of beta(4b) into and out of the nucleus indicates a specific role of this beta subunit in neurons, in communicating the activity of calcium channels to the nucleus.

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.

Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Ca(v)1.3 voltage-gated L-type Ca(2+) channels. Their voltage- and Ca(2+)-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2alpha (RIM2alpha) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Ca(v)1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the beta-subunit of the channel complex and RIM-induced slowing of both Ca(2+)- and voltage-dependent inactivation of Ca(v)1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Ca(v)1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Ca(v)1.3 gating kinetics in immature IHCs.