Neuronal Voltage-dependent Calcium Channels Research Articles

Microglial IL-10 and β-endorphin expression mediates gabapentinoids antineuropathic pain

Gabapentinoids are recommended first-line treatments for neuropathic pain. They are neuronal voltage-dependent calcium channel α2δ-1 subunit ligands and have been suggested to attenuate neuropathic pain via interaction with neuronal α2δ-1 subunit. However, the current study revealed their microglial mechanisms underlying antineuropathic pain. Intrathecal injection of gabapentin, pregabalin and mirogabalin rapidly inhibited mechanical allodynia and thermal hyperalgesia, with projected ED50 values of 30.3, 6.2 and 1.5 µg (or 176.9, 38.9 and 7.2 nmol) and Emax values of 66%, 61% and 65% MPE respectively for mechanical allodynia. Intrathecal gabapentinoids stimulated spinal mRNA and protein expression of IL-10 and β-endorphin (but not dynorphin A) in neuropathic rats with the time point parallel to their inhibition of allodynia, which was observed in microglia but not astrocytes or neurons in spinal dorsal horns by using double immunofluorescence staining. Intrathecal gabapentin alleviated pain hypersensitivity in male/female neuropathic but not male sham rats, whereas it increased expression of spinal IL-10 and β-endorphin in male/female neuropathic and male sham rats. Treatment with gabapentin, pregabalin and mirogabalin specifically upregulated IL-10 and β-endorphin mRNA and protein expression in primary spinal microglial but not astrocytic or neuronal cells, with EC50 values of 41.3, 11.5 and 2.5 µM and 34.7, 13.3 and 2.8 µM respectively. Pretreatment with intrathecal microglial metabolic inhibitor minocycline, IL-10 antibody, β-endorphin antiserum or μ-opioid receptor antagonist CTAP (but not κ- or δ-opioid receptor antagonists) suppressed spinal gabapentinoids-inhibited mechanical allodynia. Immunofluorescence staining exhibited specific α2δ-1 expression in neurons but not microglia or astrocytes in the spinal dorsal horns or cultured primary spinal cells. Thus the results illustrate that gabapentinoids alleviate neuropathic pain through stimulating expression of spinal microglial IL-10 and consequent β-endorphin.

Abstract 510: Neuronal Voltage-Dependent Calcium Channel 2.1 RNA Increased in Animal Models With Contrast-Induced Nephropathy

Objectives: Contrast media has been widely used in imaging studies and endovascular procedures. However, contrast induced nephropathy (CIN) has been a major concern in the patients undergoing angiography and less invasive endovascular surgery. The current available main test for the diagnosis of CIN is to measure serum creatinine (Cr); however, It is non-specific and maybe due to underline renal disease. The objectives of this study were to analyze the changes of different proteins in CIN animal models, and to discover potential biomarker(s) for the early diagnosis of CIN. Materials & Methods: C57B1/6J mice were used to create CIN model. A prostaglandin synthesis inhibitor (indomethacin, 10 mg/kg) and a nitric oxide synthase inhibitor (Nω-Nitro-L-arginine methyl ester, L-NAME, 10mg/kg) were injected intraperitoneally (ip) before giving Iodixanol, a commonly-used intravenous contrast, (320 mg iodine/ml, 4.9 g iodine/kg ip). Sham mice received ip injections of normal saline instead of Iodixanol. Mice kidneys were harvested at day1, day2 and day3 in different groups. RNA was extracted from half of the kidney specimen. qRT -PCR were used to quantify Kidney Injury Molecule-1 (KIM-1), neuronal voltage-dependent calcium channel 2.1 (CaV 2.1) and Cystatin C RNA expression, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as endogenous control. The other half of the specimen was fixed with formalin, embedded in paraffin, and then stained with Hematoxylin and Eosin (H&E) for histopathology evaluation. Results: H&E staining demonstrated histological damages in Iodixanol-treated groups but not in controls. KIM-1 and Cystatin C expressions were not increased after receiving Iodixanol. CaV 2.1 RNA levels were significantly increased on day 3 in Iodixanol group (p < 0.05), which was consistent with the contrast-induced damage based on histological examination. CaV 2.1 levels were also elevated on Day1 and Day 2 following iodixanol administration, although not statistically significant. Conclusions: Cav 2.1 gene expression is significantly increased in CIN animal models. Cav 2.1 may be involved in the mechanisms of CIN development, and could be a potential biomarker for the diagnosis of CIN. Further clinical investigation is needed.

Inhibition of dendritic Ca2+ spikes by GABAB receptors in cortical pyramidal neurons is mediated by a direct Gi/o‐βγ‐subunit interaction with Cav1 channels

Voltage-dependent calcium channels (VDCCs) serve a wide range of physiological functions and their activity is modulated by different neurotransmitter systems. GABAergic inhibition of VDCCs in neurons has an important impact in controlling transmitter release, neuronal plasticity, gene expression and neuronal excitability. We investigated the molecular signalling mechanisms by which GABA(B) receptors inhibit calcium-mediated electrogenesis (Ca(2+) spikes) in the distal apical dendrite of cortical layer 5 pyramidal neurons. Ca(2+) spikes are the basis of coincidence detection and signal amplification of distal tuft synaptic inputs characteristic for the computational function of cortical pyramidal neurons. By combining dendritic whole-cell recordings with two-photon fluorescence Ca(2+) imaging we found that all subtypes of VDCCs were present in the Ca(2+) spike initiation zone, but that they contribute differently to the initiation and sustaining of dendritic Ca(2+) spikes. Particularly, Ca(v)1 VDCCs are the most abundant VDCC present in this dendritic compartment and they generated the sustained plateau potential characteristic for the Ca(2+) spike. Activation of GABA(B) receptors specifically inhibited Ca(v)1 channels. This inhibition of L-type Ca(2+) currents was transiently relieved by strong depolarization but did not depend on protein kinase activity. Therefore, our findings suggest a novel membrane-delimited interaction of the G(i/o)-βγ-subunit with Ca(v)1 channels identifying this mechanism as the general pathway of GABA(B) receptor-mediated inhibition of VDCCs. Furthermore, the characterization of the contribution of the different VDCCs to the generation of the Ca(2+) spike provides new insights into the molecular mechanism of dendritic computation.

Effect of RIM 1 on inactivation of neuronal voltage-dependent calcium channels

Effect of RIM 1 on inactivation of neuronal voltage-dependent calcium channels

G Protein-induced Trafficking of Voltage-dependent Calcium Channels

Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.

The Construction of the Chiral Quaternary Carbon in the Drug Discovery Directed Towards the Novel Neuroprotective Agent E2050

AbstractFor Abstract see ChemInform Abstract in Full Text.

Discovery of novel neuronal voltage-Dependent calcium channel blockers based on emopamil left hand as a bioactive template

A series of novel neuronal voltage-dependent calcium channel (VDCC) blockers, with inhibitory activity at low micromolar and moderate solubility in water, was discovered by constructing and screening a focused library based on emopamil ( 1) left hand (ELH) as a bioactive template.

Human neuronal stargazin-like proteins, γ2, γ3 and γ4; an investigation of their specific localization in human brain and their influence on CaV2.1 voltage-dependent calcium channels expressed in Xenopus oocytes.

BackgroundStargazin (γ2) and the closely related γ3, and γ4 transmembrane proteins are part of a family of proteins that may act as both neuronal voltage-dependent calcium channel (VDCC) γ subunits and transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazoleproponinc (AMPA) receptor regulatory proteins (TARPs). In this investigation, we examined the distribution patterns of the stargazin-like proteins γ2, γ3, and γ4 in the human central nervous system (CNS). In addition, we investigated whether human γ2 or γ4 could modulate the electrophysiological properties of a neuronal VDCC complex transiently expressed in Xenopus oocytes.ResultsThe mRNA encoding human γ2 is highly expressed in cerebellum, cerebral cortex, hippocampus and thalamus, whereas γ3 is abundant in cerebral cortex and amygdala and γ4 in the basal ganglia. Immunohistochemical analysis of the cerebellum determined that both γ2 and γ4 are present in the molecular layer, particularly in Purkinje cell bodies and dendrites, but have an inverse expression pattern to one another in the dentate cerebellar nucleus. They are also detected in the interneurons of the granule cell layer though only γ2 is clearly detected in granule cells. The hippocampus stains for γ2 and γ4 throughout the layers of the every CA region and the dentate gyrus, whilst γ3 appears to be localized particularly to the pyramidal and granule cell bodies. When co-expressed in Xenopus oocytes with a CaV2.1/β4 VDCC complex, either in the absence or presence of an α2δ2 subunit, neither γ2 nor γ4 significantly modulated the VDCC peak current amplitude, voltage-dependence of activation or voltage-dependence of steady-state inactivation.ConclusionThe human γ2, γ3 and γ4 stargazin-like proteins are detected only in the CNS and display differential distributions among brain regions and several cell types in found in the cerebellum and hippocampus. These distribution patterns closely resemble those reported by other laboratories for the rodent orthologues of each protein. Whilst the fact that neither γ2 nor γ4 modulated the properties of a VDCC complex with which they could associate in vivo in Purkinje cells adds weight to the hypothesis that the principal role of these proteins is not as auxiliary subunits of VDCCs, it does not exclude the possibility that they play another role in VDCC function.

The Construction of the Chiral Quaternary Carbon in the Drug Discovery Directed towards the Novel Neuroprotective Agent E2050

Brain Ischemia is believed to induce neuronal damage by causing a [Ca2+] i overload and a sustained increase in the level of extracellular glutamate (Excitotoxicity theory). Based on the Excitotoxicity theory, inhibition of neuronal voltage-dependent calcium channels is expected to reduce excessive glutamate release at the presynaptic terminals, inhibit [Ca2+] i elevation in postsynaptic neurons, and protect neurons from cell death.E2050, which is derived from Verapamil, a known L-type calcium channel blocker, is a newly synthesized neuronal calcium channel blocker discovered at Eisai Tsukuba Research Laboratories. E2050 is an optically active compound in which the quaternary carbon center has a 2-isopropyl-2-phenyl-5-piperazinopentanitrile substituent. We report here two general routes for the preparation of enantiomerically pure E2050. First, we describe the preparation of the chiral 5-hydroxy-2-isopropyl-2-phenylpentanitrile which is the key intermediate by enzyme-catalyzed kinetic resolution. Second, we illustrate the practical asymmetric synthesis of E2050 by Sharpless epoxidation followed by MAD-induced rearrangement. We believe these methods are suitable for large-scale synthesis of E2050.

The role of high-threshold calcium channels in spinal neuron hyperexcitability induced by knee inflammation

This chapter discusses the neurobiology of voltage-dependent calcium channels (VDCCs) and summarizes the involvement of VDCCs in the spinal processing of nociceptive input from normal and inflamed knee joint. Neuronal VDCCs is subdivided on the basis of their electrophysiological and pharmacological properties into low voltage-activated or T-type channels and high voltage-activated (HVA) channels, a class that includes L-, N-, P/Q- and R-type channels. In general, L-type VDCCs are localized predominantly in the somata and proximal dendrites of neurons throughout the brain. N-type and P/Q-type VDCCs are localized in presynaptic terminals and dendritic shafts of neurons. To study the involvement of N-, P/Q-, and L-type VDCCs in the spinal processing of articular input, recordings were made from nociceptive neurons in the dorsal horn of anesthetized rats. VDCC blockers were administered topically onto the spinal cord at specific concentrations. The chapter discusses the effects of N-type, P/Q-type, and L-type channel antagonists on the responses of spinal cord neurons with input from the normal or inflamed knee joint. Effects of N-type and P/Q-type channel antagonists on the generation of inflammation-induced hyperexcitability of spinal cord neurons with input from the knee joint is also discussed.

Identification of Residues in the N Terminus of α1B Critical for Inhibition of the Voltage-Dependent Calcium Channel by Gβγ

To examine the role of the intracellular N terminus in the G-protein modulation of the neuronal voltage-dependent calcium channel (VDCC) alpha1B, we have pursued two routes of investigation. First, we made chimeric channels between alpha1B and alpha1C, the latter not being modulated by Gbeta gamma subunits. VDCC alpha1 subunit constructs were coexpressed with accessory alpha2delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. G-protein modulation of expressed alpha1 subunits was induced by activation of coexpressed dopamine (D2) receptors with quinpirole in oocytes, or by cotransfection of Gbeta1gamma2 subunits in COS-7 cells. For the chimeric channels, only those with the N terminus of alpha1B showed any G-protein modulation; further addition of the first transmembrane domain and I-II intracellular linker of alpha1B increased the degree of modulation. To determine the amino acids within the alpha1B N terminus, essential for G-protein modulation, we made mutations of this sequence and identified three amino acids (S48, R52, and R54) within an 11 amino acid sequence as being critical for G-protein modulation, with I49 being involved to a lesser extent. This sequence may comprise an essential part of a complex Gbeta gamma-binding site or be involved in its subsequent action.

N-type calcium channel/syntaxin/snap-25 complex probed by antibodies to II–III intracellular loop of the α1B subunit

Neuronal voltage-dependent calcium channels are integral components of cellular excitation and neurosecretion. In addition to mediating the entry of calcium across the plasma membrane, both N-type and P/Q-type voltage-dependent calcium channels have been shown to form stable complexes with synaptic vesicle and presynaptic membrane proteins, indicating a structural role for the voltage-dependent calcium channels in secretion. Recently, detailed structural analyses of N-type calcium channels have identified residues amino acids 718–963 as the site in the rat α 1B subunit that mediates binding to syntaxin, synaptosome-associated protein of 25 000 mol. wt and synaptotagmin [Sheng et al. (1996) Nature 379, 451–454]. The purpose of this study was to employ site-directed antibodies to target domains within and outside of the interaction site on the rat α 1B to probe potential binding sites for syntaxin/SNAP-25/synaptotagmin. Our results demonstrate that both antibodies employed in this study have access to their epitopes on the α 1B as evidenced by equivalent immunoprecipitation of native [ 125I]omega-conotoxin GVIA-labeled α 1B protein from CHAPS-solubilized preparations. The N-type voltage-dependent calcium channel immunoprecipitated by Ab CW14, the antibody directed to a domain outside of the synprint site, is associated with syntaxin and SNAP-25 with the recovery of these proteins, increasing in parallel to the recovery of α 1B. However, when we used the antibody raised to an epitope within the synprint site (Ab CW8) to immunoprecipitate N-type calcium channels, the α 1B was depleted of more than 65% of syntaxin and 80% of SNAP-25 when compared to the recovery of these proteins using Ab CW14. This is the first report of a defined epitope on the α 1B subunit II–III loop (amino acids 863–875) whose perturbation by a site-directed antibody influences the dissociation of SNAP-25 and syntaxin.

Antibodies against the β subunit of voltage-dependent calcium channels in Lambert–Eaton Myasthenic Syndrome

Lambert–Eaton myasthenic syndrome is an autoimmune disease that impairs neuromuscular transmission. Several studies suggest that neurotransmitter release is reduced by an immune response directed against the calcium channel complex of nerve terminals. The immunoglobulin G fractions from Lambert–Eaton myasthenic syndrome patients immunoprecipitate solubilized neuronal N- and P/Q-type channels and in certain cases brain, skeletal and cardiac muscle L-type channels [El Far O. et al. (1995) J. Neurochem. 64, 1696–1702; Lennon V. A. and Lambert E. H. (1989) Mayo Clin. Proc. 64, 1498–1504; Sher E. et al. (1989) Lancet ii, 640–643; Suenaga A. et al. (1996) Muscle Nerve 19, 1166–1168]. These channel immunoprecipitation assays are considered as useful for the diagnosis of this syndrome. In this study, we demonstrate that two predominant neuronal voltage-dependent calcium channel β subunits ( β 3 and β 4, of mol. wt 58 000) are general targets of Lambert–Eaton myasthenic syndrome autoantibodies. Of 20 disease sera tested, 55% were able to immunoprecipitate 35S-labeled β subunits. All five patients affected with small-cell lung carcinoma were positive for the β-subunit immunoprecipitation assay. Interestingly, only a fraction of the β-subunit-positive sera was also able to immunoprecipitate N- and P/Q-type channels, suggesting that several of the β-subunit epitopes are masked in native channels. In accordance with this observation, we found that several β-positive sera were able to prevent the interaction between calcium channel α 1 and β subunits in vitro. In cases where sera were able to immunoprecipitate β subunits, N- and P/Q-type channels, the immunoprecipitation of both channel types was either partially or entirely mediated by β-subunit antibodies. Our results suggest that assays based on the immunoprecipitation of β subunits can be used as an additional test to assist in the diagnosis of Lambert–Eaton myasthenic syndrome.

Identification of the Amino Terminus of Neuronal Ca2+Channel α1 Subunits α1B and α1E as an Essential Determinant of G-Protein Modulation

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.

Parallel Detection of Na,K-ATPase Alpha Subunit Isoforms by Pan-Specific Monoclonal mAb 9A7

While emphasis has been placed upon those proteins which either mediate or respond to the rapid influx of calcium following depolarization, there has been little emphasis upon those proteins which aid in the reequilibration of the membrane potential. In an effort to identify presynaptic membrane proteins implicated in neurosecretion, monoclonal antibodies were screened against proteins which cosegregated with neuronal voltage-dependent calcium channels (VDCC) following immunoprecipitation. One monoclonal antibody (mAb 9A7) identified a 110-kDa protein. Micropeptide sequencing of (i) the mAb 9A7 immunoaffinity purified antigen and (ii) the 110-kDa protein present in the neuronal (N-type) VDCC preparation (McEneryet al.,1991,Proc. Natl. Acad. Sci.88, 11095–11099) indicated identity with the alpha subunit(s) of the Na,K-ATPase. Further characterization by Western blotting, immunochemical localization, and immunoaffinity purification indicated that mAb 9A7 not only recognized the alpha3 isoform which is predominant in neuronal tissues but also identified the alpha1 and alpha2 isoforms. mAb 9A7 exhibited a wide cross-species reactivity and recognized human, rat, and mouse alpha subunit isoforms at an internal epitope. The pan-specificity of mAb 9A7 and the differential mobility of the alpha1 isoform relative to the alpha2 and alpha3 permitted parallel detection of multiple alpha isoforms. Western blot analysis of undifferentiated rat pheochromocytoma cell line (PC12) and human neuroblastoma (IMR32) cells indicated coexpression of the alpha1 and alpha3 isozymes. Upon differentiation of IMR32 cells by dibutrylyl-cAMP, a substantial increase in the alpha3 relative to the alpha1 isoform was observed. While the enrichment of total Na,K-ATPase may reflect the increased demand for ATP-dependent ion transport as IMR32 cells become more excitable, the specific increase in the alpha3 isoform suggests a unique role of this isoform during IMR32 cell differentiation.

Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of α 1, α 2 δ, and β subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation [3, 11]. Studies have shown the expression of several α 1 and β subtypes within neuronal tissue [8]. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit [15]. Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding α 1, α 2 δ and β subunits.

Immunohistochemical and in situ mRNA hybridisation techniques to determine the distribution of ion channels in human brain: a study of neuronal voltage-dependent calcium channels

The molecular, structural and functional characterisation of ion channels in the CNS forms an area of intense investigation in current brain research. For strategic and logistical reasons, rodents have historically been the species of choice for these studies. The examination of human CNS tissues generally presents the investigator with specific challenges that are often less problematic in animal studies, e.g. post-mortem delay/agonal status, and thus both the experimental design and techniques must be manipulated accordingly. Since much pharmaceutical interest is currently focused on neuronal ion channels, the examination of their expression in human brain material is of particular importance. We describe here the details of methods that we have developed and used successfully in the study of the expression of voltage-dependent calcium channels (VDCCs) in human CNS tissues [15]. Presynaptic neuronal VDCCs control neurotransmitter release [11, 8]and are important new drug targets [3]. They are composed of three subunits, α 1, β and α 2/ δ [4]and multiple gene classes of each protein have been identified [7, 12, 14]. Little is known, however, about the distribution of neuronal VDCCs in the human central nervous system, although initial studies have been performed in rat and rabbit [6, 13, 16].

Regulation of rat neuronal voltage-dependent calcium channels by endogenous p21-ras.

Influx of calcium through voltage-dependent calcium channels (VDCCs) has been implicated in the processes of cell growth and differentiation. Various signalling proteins, including nerve growth factor (NGF), p21-ras and src tyrosine kinases, have been suggested to have a role in the regulation of neuronal VDCCs. Using the whole-cell patch-clamp technique we have investigated the role of endogenous p21-ras in the regulation of VDCCs in primary cultured dorsal root ganglion (DRG) neurons obtained from neonatal rats. Neutralization of endogenous p21-ras by microinjection of p21-ras antibody (Y13-259) reduced the maximum peak barium current, I(max), whereas microinjection of oncogenic p21-K-ras increased the current. Thus, endogenous p21-ras is involved in the tonic regulation of calcium currents in these cells. Intracellular application of a phosphopeptide, Trk 490, which prevents the binding of the adaptor protein shc to the activated NGF receptor, so blocking p21-ras activation, reduced I(max). Similarly, deprivation of NGF by overnight incubation in NGF-free medium also reduced I(max). Together, these results suggest that NGF receptor tyrosine kinase activation of p21-ras is likely to be involved in the tonic regulation of VDCCs in DRG neurons. Deprivation of NGF combined with microinjection of p21-ras antibody (Y13-259), however, caused an even greater reduction of I(max). Thus, NGF activation can only partially explain the regulation of these currents by endogenous p21-ras. Src tyrosine kinases have been suggested to activate p21-ras. In DRG neurons, microinjection of purified src tyrosine kinase, pp60c-src, increased I(max) in these cells. However, co-microinjection of pp60c-src with Y13-259 antibody prevented the increase in I(max), implying that pp60c-src can also regulate calcium currents via the activation of endogenous p21-ras. Further support for the involvement of tyrosine kinases in VDCC regulation was provided by the application of the general tyrosine kinase inhibitor, genistein, which also reduced I(max). Thus, VDCCs in rat DRG neurons appear to be tonically up-regulated by endogenous p21-ras. This effect appears largely to involve NGF receptor tyrosine kinase activation of p21-ras. In addition, src tyrosine kinase may also regulate VDCCs, possibly via p21-ras.

The Intracellular Loop between Domains I and II of the B-Type Calcium Channel Confers Aspects of G-Protein Sensitivity to the E-Type Calcium Channel

Neuronal voltage-dependent calcium channels undergo inhibitory modulation by G-protein activation, generally involving both kinetic slowing and steady-state inhibition. We have shown previously that the beta-subunit of neuronal calcium channels plays an important role in this process, because when it is absent, greater receptor-mediated inhibition is observed (). We therefore hypothesized that the calcium channel beta-subunits normally may occlude G-protein-mediated inhibition. Calcium channel beta-subunits bind to the cytoplasmic loop between transmembrane domains I and II of the alpha1-subunits (). We have examined the hypothesis that this loop is involved in G-protein-mediated inhibition by making chimeras containing the I-II loop of alpha1B or alpha1A inserted into alpha1E (alpha1EBE and alpha1EAE, respectively). This strategy was adopted because alpha1B (the molecular counterpart of N-type channels) and, to a lesser extent, alpha1A (P/Q-type) are G-protein-modulated, whereas this has not been observed to any great extent for alpha1E. Although alpha1B, coexpressed with alpha2-delta and beta1b transiently expressed in COS-7 cells, showed both kinetic slowing and steady-state inhibition when recorded with GTPgammaS in the patch pipette, both of which were reversed with a depolarizing prepulse, the chimera alpha1EBE (and, to a smaller extent, alpha1EAE) showed only kinetic slowing in the presence of GTPgammaS, and this also was reversed by a depolarizing prepulse. These results indicate that the I-II loop may be the molecular substrate of kinetic slowing but that the steady-state inhibition shown by alpha1B may involve a separate site on this calcium channel.

The expression of neuronal voltage-dependent calcium channels in human cerebellum.

Little is known about the comparative distribution of voltage-dependent calcium channel subtypes in normal human brain. Previous studies in experimental animals have predominantly focused on the regional expression of single alpha 1 genes. We describe the preparation of riboprobes and antisera specific for human alpha 1A, alpha 1B and alpha 1E subunits and their application in comprehensive mapping studies of the human cerebellum. Within the cerebellar cortex, these pore forming proteins were found to have differential localisations when examined in adjacent sections. The alpha 1A and alpha 1B subunits broadly colocalised and were both present, though at apparently different levels, in the molecular, Purkinje and granule cell layers whilst alpha 1E was predominantly expressed in Purkinje cells. In the dentate nucleus, an area which has received little attention in previous studies, alpha 1A was highly expressed in regions in which Purkinje cell nerve terminals form synapses with deep cerebellar neurones.