Differential distribution of voltage-gated calcium channel alpha-2 delta (alpha2delta) subunit mRNA-containing cells in the rat central nervous system and the dorsal root ganglia

Voltage-gated calcium channels (VGCCs) play an essential role in controlling neurotransmitter release, neuronal excitability, and gene expression in the nervous system. The distribution of cells that contain mRNAs encoding the auxiliary alpha2delta-1, alpha2delta-2, and alpha2delta-3 subunits of the VGCCs in the central nervous system (CNS) and the dorsal root ganglia (DRG) was examined in rats by using in situ hybridization. Specific labeling of alpha2delta-1, alpha2delta-2, and alpha2delta-3 mRNAs appeared to be largely confined to neurons and was widely, although differentially, distributed in the brain, the spinal cord, and the DRG. Importantly, alpha2delta-2 mRNA was found to be expressed in interneurons in the cortex, the hippocampus, the striatum, and in regions that contain dense cholinergic neurons. Our results suggest that different alpha2delta subunits may exert distinctive functions in the CNS. The alpha2delta-1 subunit mRNA is localized in brain regions known to be involved in cortical processing, learning and memory, defensive behavior, neuroendocrine secretion, autonomic activation, primary sensory transmission, and general arousal. The alpha2delta-2 subunit mRNA is present in brain regions known to modulate the overall activities of the cortex, the hippocampus, and the thalamus. The alpha2delta-2 subunit is also found in brain regions known to be involved in olfaction, somatic motor control, fluid homeostasis, ingestive and defensive behaviors, neuroendocrine functions, and circadian rhythm. In addition to being localized in brain regions that express alpha2delta-1 and alpha2delta-2 subunit mRNAs, alpha2delta-3 subunit mRNA is highly expressed in regions involved in auditory information processing and somatic movement.     

GABA(A) receptor changes in delta subunit-deficient mice: altered expression of alpha4 and gamma2 subunits in the forebrain

The delta subunit is a novel subunit of the pentameric gamma-aminobutyric acid (GABA)(A) receptor that conveys special pharmacological and functional properties to recombinant receptors and may be particularly important in mediating tonic inhibition. Mice that lack the delta subunit have been produced by gene-targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABA(A) receptors were limited to deletion of the delta subunit or whether alterations in other GABA(A) receptor subunits were also present in the delta subunit knockout (delta-/-) mice. Immunohistochemical studies of wild-type mice confirmed the restricted distribution of the delta subunit in the forebrain. Regions with moderate to high levels of delta subunit expression included thalamic relay nuclei, caudate-putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no delta subunit labeling was evident in adjacent regions, such as the thalamic reticular nucleus, hypothalamus, and globus pallidus. Comparisons of the expression of other subunits in delta-/- and wild-type mice demonstrated substantial changes in the alpha4 and gamma2 subunits of the GABA(A) receptor in the delta-/- mice. gamma2 Subunit expression was increased, whereas alpha4 subunit expression was decreased in delta-/- mice. Importantly, alterations of both the alpha4 and the gamma2 subunits were confined primarily to brain regions that normally expressed the delta subunit. This suggests that the additional subunit changes are directly linked to loss of the delta subunit and could reflect local changes in subunit composition and function of GABA(A) receptors in delta-/- mice.     

Distribution of the voltage-dependent calcium channel alpha1G subunit mRNA and protein throughout the mature rat brain.

The molecular identity of a gene which encodes the pore-forming subunit (alpha1G) of a member of the family of low-voltage-activated, T-type, voltage-dependent calcium channels has been described recently. Although northern mRNA analyses have shown alpha1G to be expressed predominantly in the brain, the detailed cellular distribution of this protein in the central nervous system (CNS) has not yet been reported. The current study describes the preparation of a subunit specific alpha1G riboprobe and antiserum which have been used in parallel in situ mRNA hybridization and immunohistochemical studies to localize alpha1G in the mature rat brain. Both alpha1G mRNA and protein were widely distributed throughout the brain, but variations were observed in the relative level of expression in discrete nuclei. Immunoreactivity for alpha1G was typically localized in both the soma and dendrites of many neurons. Whilst alpha1G protein and mRNA expression were often observed in cells known to exhibit T-type current activity, some was also noted in regions, e.g. cerebellar granule cells, in which T-type activity has not been described. These observations may reflect differences between the subcellular distribution of channels that can be identified by immunohistochemical methods compared with electrophysiological techniques.     

Selective decrease in NR1 subunit splice variant mRNA in the hippocampus of Pb2+-exposed rats: implications for synaptic targeting and cell surface expression of NMDAR complexes

We have previously shown that exposure to environmentally relevant levels of Pb(2+) during brain development decreases the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) and NR2A genes in the hippocampus of young adult rats and was associated with deficits in hippocampal LTP and spatial learning [Neuroscience 99 (2000) 233-242]. In the present study, we demonstrate that the lower levels of NR1 subunit mRNA expressed in the Pb(2+)-exposed hippocampus are principally due to decreased levels of the NR1-4 and NR1-2 splice variants. These changes were present in the absence of changes in GluR1, PSD-95 and alphaCaMKII gene expression. A unique characteristic of these splice variants is that they lack the C1 cassette. Further, these splice variants have been shown to impart the highest cell surface expression, PKC potentiation and calcium kinetics to NMDAR complexes. Our present findings indicate that Pb(2+)-induced changes in NR1 subunit splice variant mRNA expression in the hippocampus may provide a mechanism by which Pb(2+)-exposure can modify NMDAR-mediated calcium signaling and influence the degree of synaptic plasticity.     

Expression of mRNA encoding alpha1E and alpha1G subunit in the brain of a rat model of absence epilepsy

Low voltage-activated calcium channels are thought to play a key role in the generation of spike and waves discharges characteristic of absence epilepsy. Therefore, the expression level of mRNA encoding calcium channel alpha1E and alpha1G subunits was measured in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Using quantitative RT-PCR and in situ hybridization, no difference was found in alpha1G mRNA expression between GAERS and control animals, while a decreased expression of alpha1E was seen in the cerebellum and the brain stem of the GAERS. This phenomenon was not observed in young animals when the epileptic phenotype is not expressed.     

Low-voltage-activated calcium channel subunit expression in a genetic model of absence epilepsy in the rat

The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbred strain of rats that display many of the characteristics of human absence epilepsy. In these rats, reciprocal thalamocortical projections play a critical role in the generation of spike-and-wave discharges that characterize absence seizures. When compared to those of the non-epileptic control strain, juvenile animals of the GAERS strain reportedly possess higher-amplitude T-type calcium currents in neurons of the thalamic reticular nucleus (nRt). We hypothesized that differences in calcium currents seen between GAERS and controls result from differences in expression of genes for low-voltage-activated calcium channels. Quantitative in situ hybridization was used to compare expression of alpha1G, alpha1H, alpha1I, and alpha1E calcium channel subunit mRNAs from adult and juvenile animals of the two strains. We found higher levels of alpha1H mRNA expression in nRt neurons of juvenile animals (34.9+/-2. 3 vs. 28.4+/-1.8 grains/10(3) pixels, p<0.05), perhaps accounting in part for earlier reports of elevated T-type current amplitude in those cells. In adult GAERS animals, we found elevated levels of alpha1G mRNA in neurons of the ventral posterior thalamic relay nuclei (64.8+/-3.5 vs. 53.5+/-1.7 grains/10(3) pixels, p<0.05), as well as higher levels of alpha1H mRNA in nRt neurons (32.6+/-0.8 vs. 28.2+/-1.6 grains/10(3) pixels, p<0.05). These results suggest that the epileptic phenotype apparent in adult GAERS may result in part from these significant, albeit small ( approximately 15-25%), elevations in T-type calcium channel mRNA levels.     

beta subunit reshuffling modifies N- and P/Q-type Ca2+ channel subunit compositions in lethargic mouse brain.

Neuronal voltage-dependent Ca2+ channels are heteromultimers of alpha1, beta, and alpha2delta subunits, and any one of five alpha1 subunits (alpha1A-E) may associate with one of four beta subunits (beta1-4). The specific alpha1-beta combination assembled determines single-channel properties, while variation in the proportion of each combination contributes to the functional diversity of neurons. The mouse mutant lethargic (lh) exhibits severe neurological defects due to a mutation that deletes the alpha1 subunit interaction domain of the beta4 subunit. Since beta subunits regulate critical alpha1 subunit properties in heterologous expression systems, loss of beta4 in lethargic could dramatically alter channel localization and behavior unless beta1-3 subunits can be used as substitutes in vivo. Here we demonstrate increased steady-state associations of alpha1A and alpha1B with the remaining beta1-3 subunits, without significant changes in beta1-3 mRNA abundance. The immunolocalization of alpha1A and alpha1B protein in lethargic brain is indistinguishable from wild-type by light microscopy. Furthermore, the measurement of large-amplitude P-type currents in dissociated lethargic Purkinje neurons indicates that these alpha1A-containing channels retain regulation by beta subunits. We conclude that several properties of alpha1A and alpha1B proteins are not uniquely regulated by beta4 in vivo and may be rescued by beta1-3 subunit reshuffling. The complex neurological manifestation of the lethargic mutation therefore emerges from loss of beta4 coupled with the widespread pairing of surrogate beta subunits with multiple Ca2+ channel subtypes. The existence of beta subunit reshuffling demonstrates that molecular plasticity of Ca2+ channel assembly, a normal feature of early brain development, is retained in the mature brain.     

Role of the synprint site in presynaptic targeting of the calcium channel CaV2.2 in hippocampal neurons

Sequences in the cytoplasmic II-III loop of CaV2 voltage-gated calcium channels, termed the synaptic protein interaction (synprint) site, are considered important for the functional incorporation of presynaptic calcium channels into the synaptic vesicle fusion apparatus. Two novel CaV2.2 splice variants lack large parts of the cytoplasmic II-III loop (Delta1 R756-L1139, Delta2 K737-A1001) including the synprint protein-protein interaction domain. Here we expressed green fluorescent protein (GFP)-alpha1B subunit fusion constructs of CaV2.2 splice variants in mouse hippocampal neurons to study their distribution in distinct neuronal compartments and to address the question of whether and how the synprint site functions in the presynaptic targeting of N-type calcium channels. Similar to full-length GFP-alpha1B but divergent from the somatodendritic alpha1C-HA (CaV1.2) channel type, the splice variants GFP-alpha1B-Delta1 and GFP-alpha1B-Delta2 were targeted into the axons. Nevertheless, their ability to form bona fide presynaptic clusters was almost abolished for GFP-alpha1B-Delta1 and significantly reduced for GFP-alpha1B-Delta2. Thus, the synprint site is important for normal synaptic targeting of CaV2.2 but not essential. Conversely, insertion of the synprint site into the II-III loop of alpha1C-HA did not restore axonal targeting or synaptic clustering. Together these results indicate that protein-protein interactions with the synprint site must cooperate with other targeting mechanisms in the incorporation of CaV2.2 into presynaptic specializations of hippocampal neurons but are neither necessary nor sufficient for axonal targeting. The unique targeting properties of the splice variants lacking the synprint site are suggestive of specific functions of these calcium channels apart from activating fast synaptic transmission.     

Role of Pur alpha in targeting mRNA to sites of translation in hippocampal neuronal dendrites

Using genetic inactivation in the mouse, PURA, encoding Pur alpha, is demonstrated to be essential for developmentally-timed dendrite formation in the cerebellum and hippocampus. Comparison of RNA species bound by Pur alpha prompts the hypothesis that Pur alpha functions with non-coding RNA in transport of certain mRNA molecules to sites of translation in dendrites. Pur alpha binds to human BC200 RNA, implicated in dendritic targeting, and this has homologies to 7SL RNA, implicated in compartmentalized translation. Results using hippocampal rat neurons in situ show that Pur alpha binds to BC1 RNA, implicated in dendritic targeting as a mouse counterpart of BC200, and to mRNA molecules translated in dendrites; Pur alpha is specifically located in dendrites, where it is colocalized with Map2, but not in axons, where it fails to colocalize with Ankyrin G. Pur alpha and Staufen are colocalized at dendritic sites of mRNA translation. Microtubule disruptors inhibit Pur alpha dendritic targeting and allow its mislocalization to axons. Using mouse brain, double-RNA immunoprecipitation places Pur alpha together with Staufen or FMRP on BC1 RNA and specific mRNA species in vivo. These results help define a mechanism by which Pur alpha targets specific mRNA molecules to sites of dendritic translation.     

Regional distribution of cells expressing glycine receptor alpha 2 subunit mRNA in the rat brain.

The alpha 2 subunit of the glycine receptor is expressed transiently in the rat brain during early development suggesting that this subunit may be replaced by the alpha 1 subunit in the adult brain. The expression of glycine receptor alpha 2 subunit mRNA was investigated in the 7-day-old rat brain by in situ hybridization histochemistry using oligonucleotide probes specific for this subunit. Neurons expressing alpha 2 subunit mRNA were found to be widely and abundantly distributed throughout brain. We compared the distribution of neurons expressing alpha 2 subunit mRNA with that of neurons expressing alpha 1 or beta subunit mRNA. In the lower brainstem, the location of the neurons expressing alpha 2 subunit mRNA was very similar to that of the neurons with alpha 1 or beta subunit mRNA. Neurons expressing beta subunit mRNA were widespread and numerous in the forebrain, where neurons with alpha 1 subunit mRNA were uncommon. The locations of the neurons labeled by the alpha 2 probe were very similar to those of the cells labeled by the beta probe. These findings suggest that the alpha 2 subunit is not only expressed by immature neurons containing the alpha 1 subunit, but is also common to most immature neurons having the glycine receptor. However, it should be noted that several neurons contained beta and/or alpha 1 subunit mRNA but lacked alpha 2 subunit mRNA, suggesting that the glycine receptor is heterogeneous in its composition during brain development.     

Alternative splicing of a short cassette exon in alpha1B generates functionally distinct N-type calcium channels in central and peripheral neurons.

The N-type Ca channel alpha1B subunit is localized to synapses throughout the nervous system and couples excitation to release of neurotransmitters. In a previous study, two functionally distinct variants of the alpha1B subunit were identified, rnalpha1B-b and rnalpha1B-d, that differ at two loci;four amino acids [SerPheMetGly (SFMG)] in IIIS3-S4 and two amino acids [GluThr (ET)] in IVS3-S4. These variants are reciprocally expressed in rat brain and sympathetic ganglia (). We now show that the slower activation kinetics of rnalpha1B-b (DeltaSFMG/+ET) compared with rnalpha1B-d (+SFMG/DeltaET) channels are fully accounted for by the insertion of ET in IVS3-S4 and not by the lack of SFMG in IIIS3-S4. We also show that the inactivation kinetics of these two variants are indistinguishable. Through genomic analysis we identify a six-base cassette exon that encodes the ET site and with ribonuclease protection assays demonstrate that the expression of this mini-exon is essentially restricted to alpha1B RNAs of peripheral neurons. We also show evidence for regulated alternative splicing of a six-base exon encoding NP in the IVS3-S4 linker of the closely related alpha1A gene and establish that residues NP can functionally substitute for ET in domain IVS3-S4 of alpha1B. The selective expression of functionally distinct Ca channel splice variants of alpha1B and alpha1A subunits in different regions of the nervous system adds a new dimension of diversity to voltage-dependent Ca signaling in neurons that may be important for optimizing action potential-dependent transmitter release at different synapses.     

Molecular basis for the inactivation of Ca2+- and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells.

Large-conductance Ca2+- and voltage-dependent potassium (BK) channels exhibit functional diversity not explained by known splice variants of the single Slo alpha-subunit. Here we describe an accessory subunit (beta3) with homology to other beta-subunits of BK channels that confers inactivation when it is coexpressed with Slo. Message encoding the beta3 subunit is found in rat insulinoma tumor (RINm5f) cells and adrenal chromaffin cells, both of which express inactivating BK channels. Channels resulting from coexpression of Slo alpha and beta3 subunits exhibit properties characteristic of native inactivating BK channels. Inactivation involves multiple cytosolic, trypsin-sensitive domains. The time constant of inactivation reaches a limiting value approximately 25-30 msec at Ca2+ of 10 microM and positive activation potentials. Unlike Shaker N-terminal inactivation, but like native inactivating BK channels, a cytosolic channel blocker does not compete with the native inactivation process. Finally, the beta3 subunit confers a reduced sensitivity to charybdotoxin, as seen with native inactivating BK channels. Inactivation arises from the N terminal of the beta3 subunit. Removal of the beta3 N terminal (33 amino acids) abolishes inactivation, whereas the addition of the beta3 N terminal onto the beta1 subunit confers inactivation. The beta3 subunit shares with the beta1 subunit an ability to shift the range of voltages over which channels are activated at a given Ca2+. Thus, the beta-subunit family of BK channels regulates a number of critical aspects of BK channel phenotype, including inactivation and apparent Ca2+ sensitivity.     

Developmental changes in distribution of NMDA receptor channel subunit mRNAs.

In situ hybridization analyses have revealed drastic changes in expression and distribution of five subunit mRNAs of the mouse NMDA receptor channel during brain development. The epsilon 1 subunit mRNA is expressed postnatally and widely in the brain. On the other hand, the epsilon 2 subunit mRNA is found throughout the entire embryonic brain, but its expression becomes restricted to the forebrain at postnatal stages. The epsilon 3 subunit mRNA appears postnatally and predominantly in the cerebellum, whereas the epsilon 4 subunit mRNA is abundantly expressed in the diencephalon and the brainstem at embryonic and neonatal stages. In contrast, the zeta 1 subunit mRNA distributes ubiquitously in the brain throughout development. These findings suggest that changes in the subunit composition of the NMDA receptor channel take place during brain development.     

The P/Q-type voltage-dependent calcium channel: a therapeutic target in spinocerebellar ataxia type 6

BACKGROUND: Voltage-dependent calcium channels (VDCCs) are heteromultimeric complexes that mediate calcium influx into cells; the alpha 1A subunit is the pore-forming subunit specific to the neuronal P/Q-type VDCCs. Spinocerebellar ataxia type 6 (SCA 6) is caused by an abnormal expansion of a CAG repeat in CACNA1A, which encodes the alpha 1A subunit. Heterologous expression of mutated alpha 1A subunits resulted in increased channel inactivation in electrophysiological tests. Gabapentin and pregabalin interact with the alpha 2 delta subunit of the VDCCs and improved ataxia in cases of cortical cerebellar atrophy (CCA) and ataxia-telangiectasia. MATERIALS AND METHODS: A bibliographical review was performed in order to find out if gabapentin and pregabalin could prove useful in the treatment of SCA 6. RESULTS: Gabapentin and pregabalin slowed the rate of inactivation in recombinant P/Q-type VDCCs. SCA 6 shares neuropathological findings with CCA. CONCLUSIONS: On the basis of the neuropathological identity of SCA 6 with CCA, and of the effect of gabapentin and pregabalin on recombinant VDCCs the authors put forward the hypothesis that these drugs might prove beneficial in SCA 6, as the ataxia would be expected to improve. The authors hope that researchers working with this illness will be encouraged to undertake the appropriate clinical and experimental work.     

Rat dorsal root ganglia express distinctive forms of the alpha2 calcium channel subunit

Calcium channel alpha2 delta subunit is a glycosylated structural subunit consistent of the alpha2 subunit and the delta peptide. Previous studies have indicated distinctive alpha2 subunit expression in rat spinal cord and dorsal root ganglia (DRG). This study examined whether differential glycosylation underlies the molecular basis of distinct alpha2 delta subunits. The migration patterns of deglycosylated alpha2 subunits from rat spinal cord, DRG, brain and skeletal muscle were compared in Western blots. The data reported indicate that there are two forms of the alpha2 subunit in DRG that are different from the alpha2 subunit in other tissues examined, at least at the glycosylation level. Thus, post-translational modification may be important in tissue specific and functional expression of the alpha2 delta subunit.     

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.     

The effects of aging on different C-terminal splice forms of the zeta1(NR1) subunit of the N-methyl-d-aspartate receptor in mice

Changes in NMDA receptors in the prefrontal/frontal cortex and hippocampus of C57BL/6 mice during aging show a relationship to declines in spatial learning. The present study was designed to determine whether aging influences the mRNA expression of different splice forms of the zeta1 subunit of the NMDA receptor. We examined the mRNA of 4 C-terminal splice forms with the use of in situ hybridization. The zeta1-1 splice form (+C1 and +C2 cassettes) overall showed a maintenance of mRNA density from 3 to 10 months of age, followed by a significant decline by 26 months of age. In contrast, the mRNA for the zeta1-3 splice form [+C1 and +C2'(-C2)] showed significant declines between 3- and 10-month-old mice. These declines were maintained in the old mice. The zeta1-2 splice form (-C1 and +C2) showed a near-significant decrease in expression during aging across all brain regions. The zeta1-4 subunit mRNA [-C1 and +C2' (-C2)] showed no significant changes with increased age. These results indicate that there is a differential effect of aging on different splice variants of the zeta1 subunit of the NMDA receptor and those that are affected show a different temporal pattern of aging. This heterogeneity has implications for producing imbalances in the modulation of the remaining receptors.