Expression pattern of voltage-dependent calcium channel subunits in hippocampal inhibitory neurons in mice

Different subtypes of voltage-dependent calcium channels (VDCCs) generate various types of calcium currents that play important role in neurotransmitter release, membrane excitability, calcium transients and gene expression. Well-established differences in the physiological properties and variable sensitivity of hippocampal GABAergic inhibitory neurons to excitotoxic insults suggest that the calcium homeostasis, thus VDCC subunits expression pattern is likely different in subclasses of inhibitory cells. Using double-immunohistochemistry, here we report that in mice: 1) Cav2.1 and Cav3.1 subunits are expressed in almost all inhibitory neurons; 2) subunits responsible for the L-type calcium current (Cav1.2 and Cav1.3) are infrequently co-localized with calretinin inhibitory cell marker while Cav1.3 subunit, at least in part, tends to compensate for the low expression of Cav1.2 subunit in parvalbumin-, metabotropic glutamate receptor 1alpha- and somatostatin-immunopositive inhibitory neurons; 3) Cav2.2 subunit is expressed in the majority of inhibitory neurons except in calbindin-reactive inhibitory cells; 4) Cav2.3 subunit is expressed in the vast majority of the inhibitory cells except in parvalbumin- and calretinin-immunoreactive neurons where the proportion of expression of this subunit is considerably lower. These data indicate that VDCC subunits are differentially expressed in hippocampal GABAergic interneurons, which could explain the diversity in their electrophysiological properties, the existence of synaptic plasticity in certain inhibitory neurons and their vulnerability to stressful stimuli.     

Cholinergic and dopaminergic amacrine cells differentially express calcium channel subunits in the rat retina

Immunofluorescence labeling was performed to study the expression of high voltage-activated Ca(2+) channel subunits on rat retinal cholinergic and dopaminergic amacrine cells, which were double labeled with antibodies against choline acetyltransferase and tyrosine hydroxylase, respectively. The alpha(1A) subunit was predominantly expressed on the processes but not on the somata of cholinergic amacrine cells, whereas staining for alpha(1B) and alpha(1E) was observed in both structures of the cells. Immunoreactivity of alpha(1C) and alpha(1D) was not found in the cholinergic amacrine cells. Dopaminergic amacrine cells, on the other hand, exhibited a differential expression pattern of the Ca(2+) channel subunits, with alpha(1A), alpha(1C) and alpha(1E) being expressed on both somata and processes and alpha(1B) predominantly on the processes of the cells. No alpha(1D) labeling was seen. These results suggest that Ca(2+) channel subunits differentially expressed on the cholinergic and dopaminergic amacrine cells may endow these two cell types with different physiological properties.     

Expression of voltage-gated calcium channel subunits in rat dorsal root ganglion neurons

The ability of a series of specific Galpha carboxyl-terminal antisera, (i.e. anti-Gsalpha, anti-Gi1/2alpha, anti-Gi3alpha/Goalpha, anti-Goalpha/Gi3alpha, and anti-Gq/11alpha) to disrupt (+/-)-baclofen-stimulated high-affinity guanosine triphosphatase (GTPase) activity was explored in rat cerebral cortical membranes to identify the Galpha subunit(s) involved in gamma-aminobutyric acid(B) (GABA(B)) receptor-mediated signal transduction. Pretreatment of the membranes with the AS/7 (anti-Gi1/2alpha) antiserum inhibited GABA(B) receptor-mediated response without affecting the basal activity. The RM/1 (anti-Gsalpha) and QL (anti-Gq/11alpha) antisera failed to inhibit GABA(B) receptor-coupled responses. The results of the EC/2 (anti-Gi3alpha/Goalpha) and GO/1 (anti-Goalpha/Gi3alpha) antisera were difficult to interpret since the basal activities were influenced by these antisera. These results, in conjunction with the data in our previous reconstitution study, indicate that Gi2alpha is a main transducer of GABA(B) receptor-mediated signaling in rat cerebral cortex.     

Expression of calcium channel subunits in the normal and diseased human myocardium

We investigated the expression of ₁ and subunits of the L-type Ca²⁺ channel on the protein level in cardiac preparations from normal human heart ventricles and from the hypertrophied septum of patients with hypertrophic obstructive cardiomyopathy (HOCM). 1,4-Dihydropyridine (DHP) binding and immunorecognition by polyclonal antibodies directed against the C-terminal amino acid sequences of the ₂ and ₃ subunits were used for detection and quantification of ₁, ₂, and ₃ subunits. B_max of high-affinity DHP binding was 35±2 fmol/mg protein in HOCM and 20±2 fmol/mg protein in normal human hearts (P<0.05). In rabbit hearts the anti-₂ subunit antibody immunoprecipitated 80% of the total amount of DHP-labeled Ca²⁺ channels present in the assay. Under identical experimental conditions 25% of labeled Ca²⁺ channels were recovered in the immunoprecipitates of both normal and HOCM ventricles. A similar partial immunoprecipitation was observed in pig hearts. Immunoblot analysis demonstrated that the ₂ subunit was associated with the DHP receptor/Ca²⁺ channel in cardiac muscle of rabbit, pig, and human heart. In neither of these purified cardiac Ca²⁺ channels was the ₃ subunit isoform detected. Our results suggest that both ₁ and ₂ subunit expression is upregulated in HOCM in a coordinate manner.Abbreviations B _max Maximal number of binding sites - DHP 1,4-Dihydropyridine - HOCM Hypertrophic obstructive cardiomyopathy - NH Normal human heart     

Distribution of voltage-dependent calcium channel beta subunits in the hippocampus of patients with temporal lobe epilepsy.

Voltage-dependent Ca2+ channels constitute a major class of plasma membrane channels through which a significant amount of extracellular Ca2+ enters neuronal cells. Their pore-forming alpha1 subunits are associated with cytoplasmic regulatory beta subunits, which modify the distinct biophysical and pharmacological properties of the alpha1 subunits. Studies in animal models indicate altered expression of alpha1 and/or beta subunits in epilepsy. We have focused on the regulatory beta subunits and have analysed the immunoreactivity patterns of the beta1, beta2, beta3 and beta4 subunits in the hippocampus of patients with temporal lobe epilepsy (n = 18) compared to control specimens (n = 2). Temporal lobe epilepsy specimens were classified as Ammon's horn sclerosis (n = 9) or focal lesions without alteration of hippocampal cytoarchitecture (n = 9). Immunoreactivity for the beta subunits was observed in neuronal cell bodies, dendrites and neuropil. The beta1, beta2 and beta3 subunits were found mainly in cell bodies while the beta4 subunit was primarily localized to dendrites. Compared to the control specimens, epilepsy specimens of the Ammon's horn sclerosis and of the lesion group showed a similar beta subunit distribution, except for beta1 and beta2 staining in the Ammon's horn sclerosis group: in the severely sclerotic hippocampal subfields of these specimens, beta1 and beta2 immunoreactivity was enhanced in some of the remaining neuronal cell bodies and, in addition, strongly marked dendrites. Thus, hippocampal neurons apparently express multiple classes of beta subunits which segregate into particular subcellular domains. In addition, the enhancement of beta1 and beta2 immunoreactivity in neuronal cell bodies and the additional shift of the beta1 and beta2 subunits into the dendritic compartment in severely sclerotic hippocampal regions indicate specific changes in Ammon's horn sclerosis. Altered expression of these beta subunits may lead to increased currents carried by voltage-dependent calcium channels and to enhanced synaptic excitability.     

Altered expression of voltage-dependent calcium channel alpha(1) subunits in temporal lobe epilepsy with Ammon's horn sclerosis

Voltage-dependent calcium channels, the initial components in the calcium signalling cascade, are increasingly being recognised as relevant factors in the pathology of epilepsy. To further characterise their role in temporal lobe epilepsy associated with Ammon's horn sclerosis, we investigated the immunohistochemical distribution of five different voltage-dependent calcium channel alpha(1) subunits (alpha(1A), alpha(1B), alpha(1C), alpha(1D), alpha(1E)) in 14 hippocampal specimens of patients with Ammon's horn sclerosis in comparison with eight autopsy control cases. In epilepsy specimens an increased immunoreactivity was observed for alpha(1A), alpha(1B), alpha(1D) and alpha(1E) in the neuropil of the dentate gyrus molecular layer. Dentate gyrus granule cells and residual CA3 pyramidal neurones showed enhanced immunoreactivity for alpha(1A), while labelling of these neurones was decreased for alpha(1C). Astrocytes in Ammon's horn sclerosis specimens were strongly immunoreactive for the alpha(1C) subunit contrasting with an absent astrocytic alpha(1C) labelling in controls.Our results suggest that the expression of calcium channels in neurones and glial cells is dynamically regulated in temporal lobe epilepsy, supporting the relevance of calcium signalling pathways for this disease.     

抗大鼠心室肌细胞L-型钙通道亚单位抗体的制备与验证

目的：用人工合成的大鼠心室肌细胞L-型钙通道4个亚单位的抗原表位短肽免疫家兔，产生可识别该通道不同亚单位的特异性抗体，并进行验证。方法：从大鼠心室肌细胞L-型钙通道4个亚单位的氨基酸序列中筛选出三段短肽，进行人工合成。用化学合成的多肽与破伤风类毒素（TT）以戊二醛法连接，并以此作为抗原免疫家兔制备抗大鼠心室肌细胞L-型钙通道的免疫血清，经分离纯化获得抗体。抗体的验证通过ELISA，Western blot，免疫荧光组织化学技术进行检测。结果：所制备的抗大鼠心室肌细胞L-型钙通道α1c、β2和α2/δ俗亚单位的抗体滴度分别为1：51200—1：102400、1：1280和1：1280，3个抗体结合到大鼠心肌细胞膜蛋白的分子量分别为235、89和162kD。免疫荧光组织化学实验显示，抗体均能特异性的结合在培养大鼠心室肌细胞膜上。结论：所制备的抗大鼠心室肌细胞L-型钙通道亚单位抗体能特异性识别大鼠心室肌细胞膜上L-型钙通道的不同亚单位。     

Importance of voltage-dependent inactivation in N-type calcium channel regulation by G-proteins

Direct regulation of N-type calcium channels by G-proteins is essential to control neuronal excitability and neurotransmitter release. Binding of the dimer directly onto the channel is characterized by a marked current inhibition (“ON” effect), whereas the pore opening- and time-dependent dissociation of this complex from the channel produce a characteristic set of biophysical modifications (“OFF” effects). Although G-protein dissociation is linked to channel opening, the contribution of channel inactivation to G-protein regulation has been poorly studied. Here, the role of channel inactivation was assessed by examining time-dependent G-protein de-inhibition of Ca_v2.2 channels in the presence of various inactivation-altering β subunit constructs. G-protein activation was produced via μ-opioid receptor activation using the DAMGO agonist. Whereas the “ON” effect of G-protein regulation is independent of the type of β subunit, the “OFF” effects were critically affected by channel inactivation. Channel inactivation acts as a synergistic factor to channel activation for the speed of G-protein dissociation. However, fast inactivating channels also reduce the temporal window of opportunity for G-protein dissociation, resulting in a reduced extent of current recovery, whereas slow inactivating channels undergo a far more complete recovery from inhibition. Taken together, these results provide novel insights on the role of channel inactivation in N-type channel regulation by G-proteins and contribute to the understanding of the physiological consequence of channel inactivation in the modulation of synaptic activity by G-protein coupled receptors.     

PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane

Phosphatidylinositol 3-kinase (PI3K) has been shown to enhance native voltage-dependent calcium channel (Ca(v)) currents both in myocytes and in neurons; however, the mechanism(s) responsible for this regulation were not known. Here we show that PI3K promotes the translocation of GFP-tagged Ca(v) channels to the plasma membrane in both COS-7 cells and neurons. We show that the effect of PI3K is mediated by Akt/PKB and specifically requires Ca(v)beta(2) subunits. The mutations S574A and S574E in Ca(v)beta(2a) prevented and mimicked, respectively, the effect of PI3K/Akt-PKB, indicating that phosphorylation of Ser574 on Ca(v)beta(2a) is necessary and sufficient to promote Ca(v) channel trafficking.     

The critical role of voltage-dependent calcium channel in axonal repair following mechanical trauma

Membrane disruption following mechanical injury likely plays a critical role in the pathology of spinal cord trauma. It is known that intracellular calcium is a key factor that is essential to membrane resealing. However, the differential role of calcium influx through the injury site and through voltage dependent calcium channels (VDCC) has not been examined in detail. Using a well-established ex vivo guinea-pig spinal cord white matter preparation, we have found that axonal membrane resealing was significantly inhibited following transection or compression in the presence of cadmium, a non-specific calcium channel blocker, or nimodipine, a specific L-type calcium channel blocker. Membrane resealing was assessed by the changes of membrane potential and compound action potential (CAP), and exclusion of horseradish peroxidase 60 min following trauma. Furthermore, 1 microM BayK 8644, a VDCC agonist, significantly enhanced membrane resealing. Interestingly, this effect was completely abolished when the concentration of BayK 8644 was increased to 30 microM. These data suggest that VDCC play a critical role in membrane resealing. Further, there is likely an appropriate range of calcium influx through VDCC which ensures effective axonal membrane resealing. Since elevated intracellular calcium has also been linked to axonal deterioration, blockage of VDCC is proposed to be a clinical treatment for various injuries. The knowledge gained in this study will likely help us better understand the role of calcium in various CNS trauma, which is critical for designing new approaches or perhaps optimizing the effectiveness of existing methods in the treatment of CNS trauma.     

Pharmacological characterization of voltage-dependent calcium currents in rat hippocampal neurons.

The kinetics and pharmacology of voltage-dependent calcium (Ca) currents in primary cultures of hippocampal neurons were studied using the whole cell clamp technique. The low voltage-activated (LVA) Ca current was activated at -50 mV and completely inactivated within 100 ms. This current was insensitive to omega-conotoxin (omega-CgTx) and to the calcium agonist Bay K 8644. The high-voltage-activated (HVA) Ca current was activated at -20 mV and inactivated incompletely during pulses of 200 ms duration. The snail toxin omega-CgTx revealed two pharmacological components of the HVA Ca current, one irreversibly blocked and the other insensitive to the toxin. Bay K 8644 had a clear agonistic action mainly on the omega-CgTx insensitive component of the HVA Ca current.     

Expression of Kir3.3 potassium channel subunits in supraependymal axons

The serotonergic system of the brainstem raphe is involved in mood control, the sleep-wake cycle, autonomic function, and stress response. The axons of certain dorsal raphe neurons form a dense serotonergic supraependymal plexus lining the brain ventricles, likely regulating ependymal metabolism and activity including ciliary movements and glucose homeostasis. In raphe neurons, serotonin exerts its function partly via 5-HT autoreceptors and G protein-gated inwardly rectifying potassium channels (Kir3/GIRK). To consider a similar mechanism in supraependymal fibres we examined immunocytochemically the distribution of Kir3 potassium channel subunits on supraependymal axons. In the present study, we show that the Kir3.3 subunit protein is expressed in raphe-derived axons at the light and electron microscopic level, but none of the other Kir3 subfamily members or the K_ATP channel subunits Kir6.1 and Kir6.2. Thus, Kir3.3 containing potassium channels may be of functional importance in autoregulation and excitability of supraependymal fibres and the complex serotonergic regulation along the parenchyma/CSF border.     

Age-related changes in the subtypes of voltage-dependent calcium channels in rat brain cortical synapses

Age-related changes in the relative contribution of voltage-dependent calcium channel (VDCC) subtypes to depolarization-induced Ca(2+) influx and in the density of VDCC subtypes in cortical synapses were investigated using synaptosomes and their membrane preparations from brain cortices of Wistar rats. The relative contribution of VDCC subtypes to Ca(2+) influx was determined by measuring the inhibition of depolarization-induced Ca(2+) influx with four VDCC subtype-specific peptide blockers. In adult rat synaptosomes, L-, N-, P- and Q-type channels accounted for 24, 32, 27 and 12% of the total Ca(2+) influx, respectively. Brain aging significantly reduced the relative contributions of N- and P-type channels and increased the contribution of the channels resistant to the four blockers used. The densities of VDCC subtypes, determined by binding experiments using radiolabeled PN200 -110, omega-conotoxin GVIA and omega-conotoxin MVIIC, were found to be significantly decreased in aged synaptic plasma membranes. On the contrary, the dissociation constants of the blockers were not changed except for PN200-110-sensitive L-type channels. These results suggest that aging alters the relative contributions of each VDCC subtype to depolarization-induced Ca(2+) influx and decreases the number of VDCCs in rat brain cortical synapses. These changes in VDCCs may lead to age-related hypofunction of synaptic neurotransmission in brain cortices.     

Voltage-dependent calcium channel CaV1.3 subunits regulate the light peak of the electroretinogram

In response to light, the mouse retinal pigment epithelium (RPE) generates a series of slow changes in potential that are referred to as the c-wave, fast oscillation (FO), and light peak (LP) of the electroretinogram (ERG). The LP is generated by a depolarization of the basolateral RPE plasma membrane by the activation of a calcium-sensitive chloride conductance. We have previously shown that the LP is reduced in both mice and rats by nimodipine, which blocks voltage-dependent calcium channels (VDCCs) and is abnormal in lethargic mice, carrying a null mutation in the calcium channel beta(4) subunit. To define the alpha(1) subunit involved in this process, we examined mice lacking Ca(V)1.3. In comparison with wild-type (WT) control littermates, LPs were reduced in Ca(V)1.3(-/-) mice. This pattern matched closely with that previously noted in lethargic mice, confirming a role for VDCCs in regulating the signaling pathway that culminates in LP generation. These abnormalities do not reflect a defect in rod photoreceptor activity, which provides the input to the RPE to generate the c-wave, FO, and LP, because ERG a-waves were comparable in WT and Ca(V)1.3(-/-) littermates. Our results identify Ca(V)1.3 as the principal pore-forming subunit of VDCCs involved in stimulating the ERG LP.     

Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K⁺ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 M Ca²⁺ cis and trans, and decreased with increasing cis [Ca²⁺]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca²⁺ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 M. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca²⁺] dependence following a single exponential at every Ca²⁺ concentration tested. Closed time histograms, at high [Ca²⁺], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115 ± 15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.     

Regional expression of L-type calcium channel subunits during cardiac development.

The contraction of cardiomyocytes is initiated by the entrance of extracellular calcium through specific calcium channels. Within the myocardium, L-type calcium channels are most abundant. In the heart, the main pore-forming subunit is the alpha1C, although there is a larger heterogeneity on auxiliary beta subunits. We have analyzed the distribution pattern of different alpha1C and beta subunits during cardiac development by immunohistochemistry. We observed homogeneous expression of alpha1C and beta subunits within the early tubular heart, whereas regional differences are observed during the late embryogenesis. beta2 and beta4 show differential expression within the embryonic myocardium. alpha1CD1 displays only a transient enhanced expression in the ventricular conduction system. In adult heart, the expression of the different calcium channel subunits analyzed is homogeneous along the entire myocardium except for alpha1CD1 that is practically undetectable. These findings suggest that beta subunits might play a major role in conferring calcium handling heterogeneity within the developing embryonic myocardium, while alpha1C subunits might contribute just transiently.     

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard "run-down" of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the "high-voltage-activated" Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150-450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)