Ameliorative effect of curcumin on altered expression of CACNA1A and GABRD in the pathogenesis of FeCl3-induced epilepsy.

The pivotal role played by ion-channel dysregulations in the pathogenesis of epilepsy has always garnered much attention. Since mutation of ion-channel proteins CACNA1A and GABRD have been associated with epilepsy, it is important to determine the post-traumatic epilepsy-associated changes in expression levels of these ion channel proteins. Additionally, curcumin is already known for its antiepileptic and neuroprotective potential in FeCl3-induced model of post-traumatic epilepsy. Thus, we investigated FeCl3-induced epilepsy mediated differential expression of CACNA1A and GABRD in the cortical region of the rat brain. Furthermore, we investigated the effect of curcumin on the expression of both proteins. For this, epilepsy was induced by intracortical FeCl3 injection (5 µl of 100 mM). Additionally, curcumin (conc. 1000 ppm; 75 mg/kg of b.wt.; for 14 and 28 days) was administered, mixed with normal food pellets. Results obtained from EEG-MUA and Morris water maze assay demonstrate the progression of epilepsy after FeCl3 injection. Additionally, western blotting and histological studies show the downregulation of CACNA1A and GABRD during epileptogenesis. It was observed that epilepsy-associated decline in learning and memory of animals might be linked with the dysregulation of both proteins. Results also demonstrated that curcumin administration ameliorated epilepsy-associated change in expression of both CACNA1A and GABRD proteins. In conclusion, the neuroprotective effect of curcumin against iron-induced epilepsy might be accompanied by the alleviated upregulation of these channel proteins.

CaV2 channel subtype expression in rat sympathetic neurons is selectively regulated by α2δ subunits.

Type two voltage gated calcium (CaV2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability. 1 Mechanisms that regulate CaV2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express CaV2.2 and CaV2.3. 2 We noted previously that heterologous expression of CaV2.1 but not CaV2.2 results in increased calcium current in SCG neurons. 3 In the present study, we extended these observations to show that both CaV2.1 and CaV2.3 expression resulted in increased calcium currents while CaV2.2 expression did not. Further, CaV2.1 could displace native CaV2.2 channels, but CaV2.3 expression could not. Heterologous expression of the individual accessory subunits α2δ-1, α2δ-2, α2δ-3, or ß4 alone failed to increase current density, suggesting that the calcium current ceiling when CaV2.2 was over-expressed was not due to lack of these subunits. Interestingly, introduction of recombinant α2δ subunits produced surprising effects on displacement of native CaV2.2 by recombinant channels. Both α2δ-1 and α2δ-2 seemed to promote CaV2.2 displacement by recombinant channel expression, while α2δ-3 appeared to protect CaV2.2 from displacement. Thus, we observe a selective prioritization of CaV channel functional expression in neurons by specific α2δ subunits. These data highlight a new function for α2δ subtypes that could shed light on subtype selectivity of CaV2 membrane expression.

Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and α-conotoxin Vc1.1 via GABAB receptor activation.

Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein-coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba(2+) currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5'-[ß-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.

Changes in the function and expression of T-type and N-type calcium channels in the rat bladder after bladder outlet obstruction.

PURPOSE: We evaluated possible changes in the function and expression of T-type and N-type Ca(2+) channels in the bladder of rats with bladder outlet obstruction. MATERIALS AND METHODS: Female Sprague Dawley® rats were divided into a group with bladder outlet obstruction created by partial urethral ligation and a sham operated group. Six weeks postoperatively we determined the mRNA expression of T-type and N-type Ca(2+) channels in the bladder, dorsal root ganglion and spinal cord. We also cystometrically investigated expression by intravenous administration of the T-Ca blocker RQ-00311610 or the N-type Ca(2+) channel blocker ω-conotoxin GVIA. We then performed in vitro functional studies of detrusor strips using these blockers. RESULTS: mRNA expression of T-type Ca(2+) channels in the bladder detrusor and mucosa layers, and the spinal cord dorsal horn, and N-type Ca(2+) channels in the whole bladder and detrusor layer, and the spinal cord dorsal horn was greater in the obstructed group than the sham operated group. In obstructed rats bladder capacity and voided volume increased after RQ-00311610 administration but the number of nonvoiding contractions decreased after ω-conotoxin GVIA administration. Detrusor strips from obstructed rats showed weaker contractile responses to electrical field stimulation, particularly in regard to the purinergic component. ω-Conotoxin GVIA suppressed electrical field stimulation induced contractions only in the detrusor of obstructed rats, especially the cholinergic component. CONCLUSIONS: Blocking T-type Ca(2+) channels increased bladder capacity while N-type Ca(2+) channel blockade inhibited nonvoiding contractions in rats with bladder outlet obstruction. Decreased bladder efferent neurotransmission occurred after bladder outlet obstruction, predominantly in its purinergic component and detrusor contractions via cholinergic neurotransmission were activated in a compensatory manner, probably via N-type Ca(2+) channel up-regulation.

Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped with various types of high-voltage activated Ca(v) channels and with IP3 receptors for intracellular Ca2+ signaling.

The interaction between mGluR1 and the calcium channel Cav2.1 preserves coupling in the presence of long Homer proteins.

Group I metabotropic glutamate receptors (mGluR1 and 5) are G protein coupled receptors that regulate neuronal activity in a number of ways. Some of the most well studied functions of group I mGluRs, such as initiation of multiple forms of mGluR-dependent long-term depression, require receptor localization near the post-synaptic density (PSD). This localization is in turn dependent on the Homer family of scaffolding proteins which bind to a small motif on the distal C-termini of mGluR1 and 5, localize the receptors near the PSD, strengthen coupling to post-synaptic effectors and simultaneously uncouple the mGluRs from extra-synaptic effectors such as voltage dependent ion channels. Here the selectivity of this uncoupling process was examined by testing the ability of Homer-2b to uncouple mGluR1 from multiple voltage dependent calcium channels including Ca(V2.2) (N-type), Ca(V3.2) (T-type), and Ca(V2.1) (P/Q-type) expressed in rat sympathetic neurons from the superior cervical ganglion (SCG). Of these, only the mGluR1-Ca(V2.1) modulatory pathway was insensitive to Homer-2b expression. Uncoupling from this channel was achieved by co-expression of an mGluR1 C-terminal protein designed to disrupt a previously described direct interaction between these two proteins, suggesting that this interaction allows incorporation of Ca(V2.1) into the mGluR1/Homer signaling complex, thereby preserving modulation in the presence of scaffolding Homer proteins. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Beta-subunits promote the expression of Ca(V)2.2 channels by reducing their proteasomal degradation.

The ß-subunits of voltage-gated calcium channels regulate their functional expression and properties. Two mechanisms have been proposed for this, an effect on gating and an enhancement of expression. With respect to the effect on expression, ß-subunits have been suggested to enhance trafficking by masking an unidentified endoplasmic reticulum (ER) retention signal. Here we have investigated whether, and how, ß-subunits affect the level of Ca(V)2.2 channels within somata and neurites of cultured sympathetic neurons. We have used YFP-Ca(V)2.2 containing a mutation (W391A), that prevents binding of ß-subunits to its I-II linker and found that expression of this channel was much reduced compared with WT CFP-Ca(V)2.2 when both were expressed in the same neuron. This effect was particularly evident in neurites and growth cones. The difference between the levels of YFP-Ca(V)2.2(W391A) and CFP-Ca(V)2.2(WT) was lost in the absence of co-expressed ß-subunits. Furthermore, the relative reduction of expression of Ca(V)2.2(W391A) compared with the WT channel was reversed by exposure to two proteasome inhibitors, MG132 and lactacystin, particularly in the somata. In further experiments in tsA-201 cells, we found that proteasome inhibition did not augment the cell surface Ca(V)2.2(W391A) level but resulted in the observation of increased ubiquitination, particularly of mutant channels. In contrast, we found no evidence for selective retention of Ca(V)2.2(W391A) in the ER, in either the soma or growth cones. In conclusion, there is a marked effect of ß-subunits on Ca(V)2.2 expression, particularly in neurites, but our results point to protection from proteasomal degradation rather than masking of an ER retention signal.

Expression of N-type calcium channels in human adrenocortical cells and their contribution to corticosteroid synthesis.

The inhibition of aldosterone activity is a useful approach for preventing the progression of cardiovascular and renal diseases in hypertensive patients. Although the results of our previous in vivo study suggested that N-type calcium channels may have a role in regulating plasma aldosterone levels, the direct relationship between N-type calcium channels and aldosterone production in adrenocortical cells has not been examined. In this study, the analysis of quantitative reverse transcription-PCR, western blotting, and immunocytological staining indicated the possible presence of N-type calcium channels in human adrenocortical cells (H295R cell line). Patch clamp analysis indicated that omega-conotoxin GVIA (CnTX), an N-type calcium channel inhibitor, suppressed voltage-dependent barium currents. During steroidogenesis, CnTX significantly reduced the transient calcium signaling induced by angiotensin II (Ang II) and partially prevented Ang II-induced aldosterone and cortisol formation with no significant influence on CYP11B2 and CYP11B1 mRNA expression. In addition, in α1B calcium channel subunits, knockdown significantly decreased Ang II-induced aldosterone formation with increments in CYP11B2 mRNA expression. We also investigated the inhibitory activities of some types of dihydropyridine calcium channel blockers (CCBs; cilnidipine: L-/N-type CCB, efonidipine: L-/T-type CCB, and nifedipine: L-type CCB), and these agents showed a dose-dependent inhibition effect on Ang II-induced aldosterone and cortisol production. Furthermore, only cilnidipine failed to suppress CYP11B1 expression in H295R cells. These results suggest that N-type calcium channels have a significant role in transducing the Ang II signal for aldosterone (and cortisol) biosynthesis, which may explain the mechanism by which N-type calcium channels regulate plasma aldosterone levels.

Non-additive potentiation of glutamate release by phorbol esters and metabotropic mGlu7 receptor in cerebrocortical nerve terminals.

We recently showed that prolonged activation of metabotropic glutamate receptor 7 (mGlu7) potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein and the subsequent hydrolysis of phosphatidylinositol (4,5)-bisphosphate. Release potentiation is independent of protein kinase C activation but it is dependent on the downstream release machinery, as reflected by the concomitant translocation of active zone Munc13-1 protein from the soluble to particulate fractions. Here we show that phorbol ester and mGlu7 receptor-dependent facilitation of neurotransmitter release is not additive, suggesting they share a common signalling mechanism. However, release potentiation is restricted to release sites that express N-type Ca(2+) channels, because phorbol ester and mGlu7 receptor-mediated release potentiation are absent in nerve terminals from mice lacking N-type Ca(2+) channels. In addition, phorbol esters but not mGlu7 receptors potentiate release at nerve terminals with P/Q-type Ca(2+) channels, although only under restricted conditions of Ca(2+) influx. The differential effect of phorbol esters at nerve terminals with either N- or P/Q-type Ca(2+) channels seems to be unrelated to the type Munc13 isoform expressed, and it is more likely dependent on other properties of the release machinery.

Different relationship of N- and P/Q-type Ca2+ channels to channel-interacting slots in controlling neurotransmission at cultured hippocampal synapses.

Synaptic transmission at CNS synapses is often mediated by joint actions of multiple Ca(2+) channel subtypes, most prominently, P/Q- and N-type. We have proposed that P/Q-type Ca(2+) channels saturate type-preferring slots at presynaptic terminals, which impose a ceiling on the synaptic efficacy of the channels. To test for analogous interactions for presynaptic N-type Ca(2+) channels, we overexpressed their pore-forming Ca(V)2.2 subunit in cultured mouse hippocampal neurons, recorded excitatory synaptic transmission from transfected cells, and dissected the contributions of N-, P/Q-, and R-type channels with subtype-specific blockers. Overexpression of Ca(V)2.2 did not increase the absolute size of the EPSC even though somatic N-type current was augmented by severalfold. Thus, the strength of neurotransmission is saturated with regard to levels of Ca(2+) channel expression for both N-type and P/Q-type channels. Overexpression of Ca(2+)-impermeable Ca(V)2.2 subunits decreased EPSC size, corroborating competition for channel slots. Striking asymmetries between N- and P/Q-type channels emerged when their relative contributions were compared with channel overexpression. Overexpressed N-type channels could competitively displace P/Q-type channels from P/Q-preferring slots and take over the role of supporting transmission. The converse was not found with overexpression of P/Q-type channels, regardless of their C-terminal domain. We interpret these findings in terms of two different kinds of presynaptic slots at excitatory synapses, one accepting N-type channels but rejecting P/Q-type (N(specific)) and the other preferring P/Q-type but also accepting N-type (PQ(preferring)). The interaction between channels and slots governs the respective contributions of multiple channel types to neurotransmission and, in turn, the ability of transmission to respond to various stimulus patterns and neuromodulators.

CCR2 receptor ligands inhibit Cav3.2 T-type calcium channels.

Monocyte chemoattractant protein-1 (MCP-1) is a cytokine known to be involved in the recruitment of monocytes to sites of injury. MCP-1 activates the chemokine (C-C motif) receptor 2 (CCR2), a seven-transmembrane helix G protein-coupled receptor that has been implicated in inflammatory pain responses. Here we show that MCP-1 mediates activation of the CCR2 receptor and inhibits coexpressed N-type calcium channels in tsA-201 cells via a voltage-dependent pathway. Moreover, MCP-1 inhibits Ca(v)3.2 calcium channels, but not other members of the Cav3 calcium channel family, with nanomolar affinity. Unlike in N-type channels, this modulation does not require CCR2 receptor activation and seems to involve a direct action of the ligand on the channel. Whole-cell T-type calcium currents in acutely dissociated dorsal root ganglia neurons are effectively inhibited by MCP-1, consistent with the notion that these cells express Ca(v)3.2. The effects of MCP-1 were eliminated by heat denaturation. Furthermore, they were sensitive to the application of the divalent metal ion chelator diethylenetriaminepentaacetic acid, suggesting the possibility that metal ions may act as a cofactor. Finally, small organic CCR2 receptor antagonists inhibit Ca(v)3.2 and other members of the T-type channel family with micromolar affinity. Our findings provide novel avenues for the design of small organic inhibitors of T-type calcium channels for the treatment of pain and other T-type channel linked disorders.

Novel splice variants of rat CaV2.1 that lack much of the synaptic protein interaction site are expressed in neuroendocrine cells.

Voltage-gated Ca(2+) channels are responsible for the activation of the Ca(2+) influx that triggers exocytotic secretion. The synaptic protein interaction (synprint) site found in the II-III loop of Ca(V)2.1 and Ca(V)2.2 mediates a physical association with synaptic proteins that may be crucial for fast neurotransmission and axonal targeting. We report here the use of nested PCR to identify two novel splice variants of rat Ca(V)2.1 that lack much of the synprint site. Furthermore, we compare immunofluorescence data derived from antibodies directed against sequences in the Ca(V)2.1 synprint site and carboxyl terminus to show that channel variants lacking a portion of the synprint site are expressed in two types of neuroendocrine cells. Immunofluorescence data also suggest that such variants are properly targeted to neuroendocrine terminals. When expressed in a mammalian cell line, both splice variants yielded Ca(2+) currents, but the variant containing the larger of the two deletions displayed a reduced current density and a marked shift in the voltage dependence of inactivation. These results have important implications for Ca(V)2.1 function and for the mechanisms of Ca(V)2.1 targeting in neurons and neuroendocrine cells.

[Neurons in the corpus callosum of rats: expression of Cav2.2 and their connection].

OBJECTIVE: To prove the existence neurons in the rat corpus callosum, the potential function of these neurons and their connection. METHODS: Immunohistochemistry was used performed to examine the expressions of NeuN, a mature neuron marker,and N-type voltage-dependent valcium channel alpha1-subunit (Cav2.2)in the section of the rat corpus callosum. Horseradish peroxidase (HRP) normal sodium solution (30%), the retrograde tracer,was injected under the frontal forceps of corpus callousm and HRP absorbed by the process of neurons in the brain slices was stained with tetramethyl benzidine. RESULTS: There were some NeuN positive cells in the rat corpus callosum and Cav2.2 was detected in some of these NeuN positive cells.Neurons with positive HRP were found in the rat corpus callosum and some of these neurons connected to the cortex or corpus striatum. CONCLUSION: There are a few neurons in the corpus callosum of adult rats and some of them express Cav2.2. Neurons in the corpus callosum have connections with the brain cortex or corpora striatum.

Molecular and immunohistochemical studies in expression of voltage-dependent Ca2+ channels in dorsal root ganglia from streptozotocin-induced diabetic mice.

We have recently demonstrated that intrathecal injection of a selective P/Q-type blocker of the voltage-dependent Ca(2+) channels (VDCCs) significantly inhibited the mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice, its antinociceptive effect being greater than in controls. In this study, to further clarify the underlying mechanism of the STZ-induced hyperalgesia, we investigated the expression level of the VDCC alpha1A and alpha1B subunits in the dorsal root ganglia (DRGs) and the dorsal spinal cord under this hyperalgesia. Real-time PCR analysis showed mRNA expression of alpha1A (P/Q-type), but not alpha1B (N-type), was significantly increased in the DRGs from the STZ-induced diabetic mice. On the other hand, gene expression of both alpha1 subunits was not changed in the dorsal part of the spinal cord. In diabetic DRG neurons, the number of large nerve cells was significantly reduced, whereas small neurons were significantly increased. Immunohistochemical study demonstrated the alpha1A-positive neurons, but not alpha1B-positive neurons, increased significantly greater in diabetic DRGs than in control in all cell size. These results indicate that an alteration in expression of P/Q-type VDCCs, especially in the small and medium-diameter primary afferent fibers, in pain pathways ascending input to the spinal cord may be involved in hypersensitivity in STZ-induced diabetes.

Interleukin-1beta regulation of N-type Ca2+ channels in cortical neurons.

Interleukin-1beta (IL-1beta) has been found to play an important role in various diseases in the central nervous system (CNS) and exhibit neuroprotective effects in some conditions. The transmitter release in brain is controlled by voltage-gated Ca(2+) channels (VGCCs), predominantly N-type Ca(2+) channels (NCCs). Although both IL-1beta and NCCs are implicated regulating excitotoxicity and Ca(2+) homeostasis, it is not known whether IL-1beta modulates NCCs directly. In present study, we examined the effects of IL-1beta treatment (10 ng/ml, 24 h) on NCCs in cultured cortical neurons using patch-clamp recording and immunoblot assay. Our results showed that IL-1beta decreased NCC currents by approximately 50%, which made up 40% of the whole-cell Ca(2+) current demonstrated by omega-conotoxin-GVIA, and also significantly downregulated the expression of NCC protein. The residual Ca(2+) currents except L-type Ca(2+) channel currents and NCC currents were not affected by IL-1beta. Our finding, IL-1beta inhibits the activity of NCC via suppressing NCC protein expression provides new insight into the neuroprotective role of IL-1beta in CNS.

Effect of Abeta exposure on the mRNA expression patterns of voltage-sensitive calcium channel alpha 1 subunits (alpha 1A-alpha 1D) in human SK-N-SH neuroblastoma.

Evidence for loss of Ca(2+) homeostasis through voltage-sensitive Ca(2+) channels (VSCCs) contribution to neuronal degeneration induced by beta-amyloid protein (Abeta) is considerable and rapidly increasing. Thus, the expression patterns of four alpha(1) subunits for P/Q (alpha(1A))-, N (alpha(1B))-, and L (alpha(1C) and alpha(1D))-type VSCCs before and after Abeta exposure were investigated in human SK-N-SH neuroblastoma. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a constitutive and abundant co-expression of mRNA for alpha(1A) and alpha(1D) subunit in control cells. The mRNA expression of another L-type subunit alpha(1C) was undetectable in control cells while N-type subunit alpha(1B) was relative lower when compared to alpha(1A) and alpha(1D) subunits. Interestingly, mRNA levels of alpha(1A), alpha(1B), and alpha(1C) were remarkably and time-dependently increased in response to Abeta (20 microM) for 72 h culture period. In contrast, the constitutively expressed alpha(1D) mRNA was not further modified during Abeta exposure. Western blot analysis of four alpha(1) subunits expression was consistent with the findings obtained by RT-PCR. In conclusion, our results suggested that P/Q-, N-, as well as L-type Ca(2+) channel genes might be existed in SK-N-SH cells. Among them, mRNA for alpha(1A), alpha(1B), and alpha(1D) were expressed constitutively while alpha(1C) were inducible. Furthermore, Abeta exposure selectively modulates the transcription of alpha(1A), alpha(1B), and alpha(1C) subunits. These suggested that except activating of existed VSCCs, up-regulation of alpha(1) subunits expression might also contribute to Abeta-induced neuronal toxicity and the complex of these VSCCs expression may participate in Ca(2+) current disturbance in Alzheimer's disease.

Expression analysis of P/Q-type Ca2+ channel alpha 1A subunit mRNA in olfactory mitral cell in N-type Ca2+ channel alpha 1B subunit gene-deficient mice.

N-type and P/Q-type Ca2+ channels play an important role in the processing of olfactory information. However, N-type Ca2+ channel alpha1B-deficient mice show normal behavior, presumably owing to compensation by other Ca2+ channels. P/Q-type Ca2+ channel alpha1A mRNA was expressed at a higher level in olfactory bulb of homozygous alpha1B-deficient mice than wild-type or heterozygous mice. LacZ expression in olfactory mitral cells of homozygous alpha1B-deficient x alpha1A1.5-lacZ mice, carrying a 1.5-kb 5'-upstream fragment of the alpha1A gene fused to the lacZ reporter gene, was increased compared to that in wild-type or heterozygous mice. Therefore, a possible explanation for the normal behavior of alpha1B-deficient mice is compensation by the alpha1A gene and that the 1.5-kb 5'-upstream region of this gene contains an enhancer cis-element for compensation in olfactory mitral cells.

Electrophysiological and molecular evidence of L-(Cav1), N- (Cav2.2), and R- (Cav2.3) type Ca2+ channels in rat cortical astrocytes.

Changes in intracellular Ca2+ levels are an important signal underlying neuron-glia cross-talk, but little is known about the possible role of voltage-gated Ca2+ channels (VGCCs) in controlling glial cell Ca2+ influx. We investigated the pharmacological and biophysical features of VGCCs in cultured rat cortical astrocytes. In whole-cell patch-clamp experiments, L-channel blockade (5 microM nifedipine) reduced Ba2+ current amplitude by 28% of controls, and further decrease (32%) was produced by N-channel blockade (3 microM omega-conotoxin-GVIA). No significant additional changes were observed after P/Q channel blockade (3 microM omega-conotoxin-MVIIC). Residual current (36% of controls) amounted to roughly the same percentage (34%) that was abolished by R-channel blockade (100 nM SNX-482). Electrophysiological evidence of L-, N-, and R-channels was associated with RT-PCR detection of mRNA transcripts for VGCC subunits alpha1C (L-type), alpha1B (N-type), and alpha1E (R-type). In cell-attached recordings, single-channel properties (L-currents: amplitude, -1.21 +/- 0.02 pA at 10 mV; slope conductance, 22.0 +/- 1.1 pS; mean open time, 5.95 +/- 0.24 ms; N-currents: amplitude, -1.09 +/- 0.02 pA at 10 mV; slope conductance, 18.0 +/- 1.1 pS; mean open time, 1.14 +/- 0.02 ms; R-currents: amplitude, -0.81 +/- 0.01 pA at 20 mV; slope conductance, 10.5 +/- 0.3 pS; mean open time, 0.88 +/- 0.02 ms) resembled those of corresponding VGCCs in neurons. These novel findings indicate that VGCC expression by cortical astrocytes may be more varied than previously thought, suggesting that these channels may indeed play substantial roles in the regulation of astrocyte Ca2+ influx, which influences neuron-glia cross-talk and numerous other calcium-mediated glial-cell functions.

Differential expression of alpha 1 and beta subunits of voltage dependent Ca2+ channel at the neuromuscular junction of normal and P/Q Ca2+ channel knockout mouse.

Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming alpha(1) and the regulatory alpha(2)delta, gamma and beta subunits. Molecular and functional studies have revealed which alpha(1) subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by alpha(1A) subunit, mediate evoked transmitter release. The generation of alpha(1A)-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs alpha(1) and beta subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The alpha(1A) subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits alpha(1B), alpha(1D) and alpha(1E) were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the alpha(1A). On the other hand, the beta(1b), beta(2a) and beta(4) were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the alpha(1A) subunit, P/Q channel under certain pathological conditions.