Altered cocaine effects in mice lacking Ca(v)2.3 (alpha(1E)) calcium channel

Much evidence indicates that calcium channel plays a role in cocaine-induced behavioral responses. We assessed the contributions of Ca(v)2.3 (alpha(1E)) calcium channel to cocaine effects using Ca(v)2.3 knockout mice (Ca(v)2.3-/-). Acute administration of cocaine enhanced the locomotor activity in wild-type mice (Ca(v)2.3+/+), but failed to produce any response in Ca(v)2.3-/- mice. Repeated exposure to cocaine induced the behavioral sensitization and conditioned place preference in both genotypes. Pretreatment with a D1-receptor antagonist, SCH23390, blocked the cocaine-induced place preference in Ca(v)2.3+/+ mice; however, it had no significant effect in Ca(v)2.3-/- mice. Microdialysis and RT-PCR analysis revealed that the levels of extracellular dopamine and dopamine D1 and D2 receptor mRNAs were not altered in Ca(v)2.3-/- mice. These data indicate that Ca(v)2.3 channel contributes to the locomotor-stimulating effect of cocaine, and the deletion of Ca(v)2.3 channel reveals the presence of a novel pathway leading to cocaine rewarding which is insensitive to D1 receptor antagonist.     

Mice lacking Ca(v)2.3 (alpha1E) calcium channel exhibit hyperglycemia.

To investigate the functional role of Ca(v)2.3 channel in glucose homeostasis, we performed in vivo glucose tolerance and insulin tolerance tests together with stress-induced glucose release tests using mice deficient in Ca(v)2.3 channel (Ca(v)2.3-/-). The Ca(v)2.3-/- mice were significantly heavier than wild-type mice. In glucose tolerance and insulin tolerance tests, Ca(v)2.3-/- mice showed a significantly higher blood glucose level compared to wild-type mice. However, stress-induced blood glucose changes in Ca(v)2.3-/- mice were similar to those in wild-type mice. These results suggest that Ca(v)2.3 channel plays a role in glucose homeostasis by reducing insulin sensitivity and that Ca(v)2.3-/- mice exhibit symptoms resembling non-insulin-dependent diabetes mellitus.     

Altered cerebellar function in mice lacking CaV2.3 Ca2+ channel

Voltage-dependent Ca(2+) channels play important roles in cerebellar functions including motor coordination and learning. Since abundant expression of Ca(V)2.3 Ca(2+) channel gene in the cerebellum was detected, we searched for possible deficits in the cerebellar functions in the Ca(V)2.3 mutant mice. Behavioral analysis detected in delayed motor learning in rotarod tests in mice heterozygous and homozygous for the Ca(V)2.3 gene disruption (Ca(V)2.3+/- and Ca(V)2.3-/-, respectively). Electrophysiological analysis of mutant mice revealed perplexing results: deficit in long-term depression (LTD) at the parallel fiber Purkinje cell synapse in Ca(V)2.3+/- mice but apparently normal LTD in Ca(V)2.3-/- mice. On the other hand, the number of spikes evoked by current injection in Purkinje cells under the current-clamp mode decreased in Ca(V)2.3 mutant mice in a gene dosage-dependent manner, suggesting that Ca(V)2.3 channel contributed to spike generation in Purkinje cells. Thus, Ca(V)2.3 channel seems to play some roles in cerebellar functions.     

Identification of the calcium channel alpha 1E (Ca(v)2.3) isoform expressed in atrial myocytes

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.     

Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1))

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.     

Role of alpha1 2.3 subunit I-II linker sites in the enhancement of Ca(v) 2.3 current by phorbol 12-myristate 13-acetate and acetyl-beta-methylcholine

Potentiation of Ca(v) 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl-beta-methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the alpha1 2.3 subunit. Mutational analysis of potential PKC sites unique to the alpha1 2.3 subunit revealed several sites in the II-III linker that are specific to MCh (Kamatchi, G., Franke, R., Lynch, C., III, and Sando, J. (2004) J. Biol. Chem. 279, 4102-4109). To identify sites responsive to PMA, Ser/Thr --> Ala mutations were made in potential PKC sites homologous to the alpha1 2.3 and 2.2 subunits, both of which respond to PMA. Wild type alpha1 2.3 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits and muscarinic M1 receptors. Inward current (I(Ba)) was recorded using Ba2+ as the charge carrier. Thr-365 of the I-II linker was identified as the primary site of PMA action, and this site also was required, along with the previously identified MCh-selective sites, for the MCh response. Ser-369 and Ser-1995 contributed to current enhancement only if Thr-365 also was available. Mutation of the essential sites to Asp increased the basal I(Ba) and caused a corresponding decrease in the PMA or MCh responses, consistent with possible regulation of these sites by phosphorylation. These results suggest that PMA and MCh both activate a pathway that can regulate the common PMA-sensitive sites in the I-II linker but that MCh also activates an additional pathway required for regulation of the MCh-unique sites, especially in the II-III linker.     

Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.     

Retrieval of context-associated memory is dependent on the Ca(v)3.2 T-type calcium channel

Among all voltage-gated calcium channels, the T-type Ca²⁺ channels encoded by the Ca_v3.2 genes are highly expressed in the hippocampus, which is associated with contextual, temporal and spatial learning and memory. However, the specific involvement of the Ca_v3.2 T-type Ca²⁺ channel in these hippocampus-dependent types of learning and memory remains unclear. To investigate the functional role of this channel in learning and memory, we subjected Ca_v3.2 homozygous and heterozygous knockout mice and their wild-type littermates to hippocampus-dependent behavioral tasks, including trace fear conditioning, the Morris water-maze and passive avoidance. The Ca_v3.2 ^−/− mice performed normally in the Morris water-maze and auditory trace fear conditioning tasks but were impaired in the context-cued trace fear conditioning, step-down and step-through passive avoidance tasks. Furthermore, long-term potentiation (LTP) could be induced for 180 minutes in hippocampal slices of WTs and Ca_v3.2 ^+/− mice, whereas LTP persisted for only 120 minutes in Ca_v3.2 ^−/− mice. To determine whether the hippocampal formation is responsible for the impaired behavioral phenotypes, we next performed experiments to knock down local function of the Ca_v3.2 T-type Ca²⁺ channel in the hippocampus. Wild-type mice infused with mibefradil, a T-type channel blocker, exhibited similar behaviors as homozygous knockouts. Taken together, our results demonstrate that retrieval of context-associated memory is dependent on the Ca_v3.2 T-type Ca²⁺ channel.     

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.     

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type)

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.     

Disturbances in glucose-tolerance, insulin-release, and stress-induced hyperglycemia upon disruption of the Ca(v)2.3 (alpha 1E) subunit of voltage-gated Ca(2+) channels

Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q, R type) coordinate Ca(2+)-dependent processes in neurons and neuroendocrine cells. Expressional and functional data have suggested a role for Ca(v)2.3 Ca(2+) channels in endocrine processes. To verify its role in vivo, Ca(v)2.3(-/-) mutant mice were generated, thus deficient in alpha 1E/R-type Ca(2+) channel. Intraperitoneal injection of D-glucose showed that glucose tolerance was markedly reduced, and insulin release into plasma was impaired in Ca(v)2.3-deficient mice. In isolated islets of Langerhans from these animals, no glucose-induced insulin release was detected. Further, in stressed Ca(v)2.3-deficient mice, the rate of glucose release into the blood was only 29% of that observed for wild-type animals. Thus, the deletion of Ca(v)2.3 causes deficits not only in insulin release but also in stress-induced hyperglycemia. The complex phenotype of Ca(v)2.3-deficient mice has dual components related to endocrine and neurological defects. The present findings provide direct evidence of a functional role for the Ca(v)2.3 subunit in hormone secretion and glucose homeostasis.     

Regulation of maximal open probability is a separable function of Ca(v)beta subunit in L-type Ca2+ channel, dependent on NH2 terminus of alpha1C (Ca(v)1.2alpha)

beta subunits (Ca(v)beta) increase macroscopic currents of voltage-dependent Ca2+ channels (VDCC) by increasing surface expression and modulating their gating, causing a leftward shift in conductance-voltage (G-V) curve and increasing the maximal open probability, P(o,max). In L-type Ca(v)1.2 channels, the Ca(v)beta-induced increase in macroscopic current crucially depends on the initial segment of the cytosolic NH2 terminus (NT) of the Ca(v)1.2alpha (alpha1C) subunit. This segment, which we term the "NT inhibitory (NTI) module," potently inhibits long-NT (cardiac) isoform of alpha1C that features an initial segment of 46 amino acid residues (aa); removal of NTI module greatly increases macroscopic currents. It is not known whether an NTI module exists in the short-NT (smooth muscle/brain type) alpha(1C) isoform with a 16-aa initial segment. We addressed this question, and the molecular mechanism of NTI module action, by expressing subunits of Ca(v)1.2 in Xenopus oocytes. NT deletions and chimeras identified aa 1-20 of the long-NT as necessary and sufficient to perform NTI module functions. Coexpression of beta2b subunit reproducibly modulated function and surface expression of alpha1C, despite the presence of measurable amounts of an endogenous Ca(v)beta in Xenopus oocytes. Coexpressed beta2b increased surface expression of alpha1C approximately twofold (as demonstrated by two independent immunohistochemical methods), shifted the G-V curve by approximately 14 mV, and increased P(o,max) 2.8-3.8-fold. Neither the surface expression of the channel without Ca(v)beta nor beta2b-induced increase in surface expression or the shift in G-V curve depended on the presence of the NTI module. In contrast, the increase in P(o,max) was completely absent in the short-NT isoform and in mutants of long-NT alpha1C lacking the NTI module. We conclude that regulation of P(o,max) is a discrete, separable function of Ca(v)beta. In Ca(v)1.2, this action of Ca(v)beta depends on NT of alpha1C and is alpha1C isoform specific.     

Effects of alcoholic beverage treatment on spatial learning and fear memory in mice

Although chronic ethanol treatment is known to impair learning and memory, humans commonly consume a range of alcoholic beverages. However, the specific effects of some alcoholic beverages on behavioral performance are largely unknown. The present study compared the effects of a range of alcoholic beverages (plain ethanol solution, red wine, sake and whiskey; with a matched alcohol concentration of 10%) on learning and memory. 6-week-old C57BL6J mice were orally administered alcohol for 7 weeks. The results revealed that red wine treatment exhibited a trend toward improvement of spatial memory and advanced extinction of fear memory. Additionally, red wine treatment significantly increased mRNA levels of brain-derived neurotrophic factor (BDNF) and N-methyl-D-aspartate (NMDA) receptors in mice hippocampus. These results support previous reports that red wine has beneficial effects.     

The C-terminus of human Ca(v)2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines

Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.     

Enhanced learning and memory in mice lacking Na+/Ca2+ exchanger 2

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) plays a role in regulation of intracellular Ca(2+) concentration via the forward mode (Ca(2+) efflux) or the reverse mode (Ca(2+) influx). To define the physiological function of the exchanger in vivo, we generated mice deficient for NCX2, the major isoform in the brain. Mutant hippocampal neurons exhibited a significantly delayed clearance of elevated Ca(2+) following depolarization. The frequency threshold for LTP and LTD in the hippocampal CA1 region was shifted to a lowered frequency in the mutant mice, thereby favoring LTP. Behaviorally, the mutant mice exhibited enhanced performance in several hippocampus-dependent learning and memory tasks. These results demonstrate that NCX2 can be a temporal regulator of Ca(2+) homeostasis and as such is essential for the control of synaptic plasticity and cognition.     

Attenuated pain responses in mice lacking Ca(V)3.2 T-type channels

Although T-type Ca(2+) channels are implicated in nociception, the function of specific subtypes has not been well defined. Here, we compared pain susceptibility in mice lacking Ca(V)3.2 subtype of T-type Ca(2+) channels (Ca(V)3.2(-/-)) with wild-type littermates in various behavioral models of pain to explore the roles of Ca(V)3.2 in the processing of noxious stimuli in vivo. In acute mechanical, thermal and chemical pain tests, Ca(V)3.2(-/-) mice showed decreased pain responses compared to wild-type mice. Ca(V)3.2(-/-) mice also displayed attenuated pain responses to tonic noxious stimuli such as intraperitoneal injections of irritant agents and intradermal injections of formalin. In spinal nerve ligation-induced neuropathic pain, however, behavioral responses of Ca(V)3.2(-/-) mice were not different from those of wild-type mice. The present study reveals that the Ca(V)3.2 subtype of T-type Ca(2+) channels are important in the peripheral processing of noxious signals, regardless of modality, duration or affected tissue type.     

Molecular and biophysical basis of glutamate and trace metal modulation of voltage-gated Ca(v)2.3 calcium channels

Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Ca(v)2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Ca(v)2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1-IS2 (H111) and IS2-IS3 (H179 and H183) loops potentiates Ca(v)2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Ca(v)2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the "ON" position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Ca(v)2.3, potentially affecting synaptic transmission and plasticity in the brain.