Known calcium channel alpha1 subunits can form low threshold small conductance channels with similarities to native T-type channels.

Native T-type voltage-dependent calcium channels are low voltage-activated and have a small single channel conductance of 5-8 pS, which distinguishes them from any known cloned calcium channels whose conductances are 12-25 pS. Here, we show that when alpha1B, alpha1E, or alpha1C are expressed in COS7 cells, which contain no endogenous calcium channel subunits or calcium channels, they each exhibit a 4-7 pS channel as well as a large conductance channel. At low depolarizations, or when the alpha1 subunit is expressed in the absence of auxiliary alpha2-delta or beta subunits, the small conductance channels are seen alone, and their biophysical properties, including voltage dependence and kinetics of activation and inactivation, are very similar to native T-type calcium channels.

Intracellular calcium regulates the survival of early sensory neurons before they become dependent on neurotrophic factors.

To investigate the role of intracellular Ca2+ in the survival of developing neurons before they become neurotrophic factor dependent, we have studied chick embryo nodose neurons, which have a particularly protracted period of neuorophic factor independence. Pharmacological reduction of intracellular free Ca2+ or depletion of either Ca(2+)-regulated or inositol trisphosphate-regulated intracellular Ca2+ stores kills early neurotrophic factor-independent neurons, but has a negligible effect on older neurons growing in the presence of brain-derived neutrotrophic factor. Shortly before they become dependent on brain-derived neurotrophic factor, nodose neurons express L-type Ca2+ channels and their survival can be enhanced by depolarization-induced Ca2+ influx. We conclude that intracellular Ca2+ plays a role in regulating neuronal survival both prior to and after the onset of neurotrophic factor dependence, but does not mediate the survival-promoting effects of neurotrophic factors.

Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels.

Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.

Facilitation of Ca2+ current in excitable cells.

Voltage-dependent Ca2+ channels are one of the main routes for the entry of Ca2+ into excitable cells. These channels are unique in cell-signalling terms in that they can transduce an electrical signal (membrane depolarization) via Ca2+ entry into a chemical signal, by virtue of the diverse range of intracellular Ca(2+)-dependent enzymes and processes. In a variety of cell types, currents through voltage-dependent Ca2+ channels can be increased in amplitude by a number of means. Although the term facilitation was originally defined as an increase of Ca2+ current resulting from one or a train of prepulses to depolarizing voltages, there is a great deal of overlap between facilitation by this means and enhancement by other routes, such as phosphorylation.

Interactions of polyamines with neuronal ion channels.

Polyamines, a group of aliphatic amines, exert selective and complex actions on a variety of ion channels. Polyamines are found endogenously, as normal metabolic intermediates, and also in the venoms of several invertebrates, where they act as potent neurotoxins. In addition, evidence suggests that polyamines may mediate or potentiate excitotoxic mechanisms responsible for neuronal damage during ischaemia. Now that the structures and functions of various polyamines are beginning to be deduced, and synthetic analogues become available, these compounds are of importance, not only as pharmacological tools to study specific receptor/ion channel complexes, but also as templates on which to base drugs designed for neuroprotective effects in a number of neurodegenerative disorders.

Identification of residues in the N terminus of alpha1B critical for inhibition of the voltage-dependent calcium channel by Gbeta gamma.

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.

Dominant-negative synthesis suppression of voltage-gated calcium channel Cav2.2 induced by truncated constructs.

Voltage-gated calcium channel alpha1 subunits consist of four domains (I-IV), each with six transmembrane segments. A number of truncated isoforms have been identified to occur as a result of alternative splicing or mutation. We have examined the functional consequences for expression of full-length Ca(v)2.2 (alpha1B) of its coexpression with truncated constructs of Ca(v)2.2. Domains I-II or domains III-IV, when expressed individually, together with the accessory subunits beta1b and alpha2delta-1, did not form functional channels. When they were coexpressed, low-density whole-cell currents and functional channels with properties similar to wild-type channels were observed. However, when domain I-II, domain III-IV, or domain I alone were coexpressed with full-length Ca(v)2.2, they markedly suppressed its functional expression, although at the single channel level, when channels were recorded, there were no differences in their biophysical properties. Furthermore, when it was coexpressed with either domain I-II or domain I, the fluorescence of green fluorescent protein (GFP)-Ca(v)2.2 and expression of Ca(v)2.2 protein was almost abolished. Suppression does not involve sequestration of the Ca(v)beta subunit, because loss of GFP-Ca(v)2.2 expression also occurred in the absence of beta subunit, and the effect of domain I-II or domain I could not be mimicked by the cytoplasmic I-II loop of Ca(v)2.2. It requires transmembrane segments, because the isolated Ca(v)2.2 N terminus did not have any effect. Our results indicate that the mechanism of suppression of Ca(v)2.2 by truncated constructs containing domain I involves inhibition of channel synthesis, which may represent a role of endogenously expressed truncated Ca(v) isoforms.

Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.

Functional expression and characterization of a voltage-gated CaV1.3 (alpha1D) calcium channel subunit from an insulin-secreting cell line.

L-type calcium channels mediate depolarization-induced calcium influx in insulin-secreting cells and are thought to be modulated by G protein-coupled receptors (GPCRs). The major fraction of L-type alpha1-subunits in pancreatic beta-cells is of the neuroendocrine subtype (CaV1.3 or alpha1D). Here we studied the biophysical properties and receptor regulation of a CaV1.3 subunit previously cloned from HIT-T15 cells. In doing so, we compared this neuroendocrine CaV1.3 channel with the cardiac L-type channel CaV1.2a (or alpha1C-a) after expression together with alpha2delta- and beta3-subunits in Xenopus oocytes. Both the current voltage relation and voltage dependence of inactivation for the neuroendocrine CaV1.3 channel were shifted to more negative potentials compared with the cardiac CaV1.2 channel. In addition, the CaV1.3 channel activated and inactivated more rapidly than the CaV1.2a channel. Both subtypes showed a similar sensitivity to the dihydropyridine (+)isradipine. More interestingly, the CaV1.3 channels were found to be stimulated by ligand-bound G(i)/G(o)-coupled GPCRs whereas a neuronal CaV2.2 (or alpha1B) channel was inhibited. The observed receptor-induced stimulation of CaV1.3 channels could be mimicked by phorbol-12-myristate-13-acetate and was sensitive to inhibitors of protein kinases, but not to the phosphoinositol-3-kinase-inhibitor wortmannin, pointing to serine/threonine kinase-dependent regulation. Taken together, we describe a neuroendocrine L-type CaV1.3 calcium channel that is stimulated by G(i)/G(o)-coupled GPCRs and differs significantly in distinct biophysical characteristics from the cardiac subtype (CaV1.2a), suggesting that the channels have different roles in native cells.

1,4-Dihydropyridines modulate GTP hydrolysis by Go in neuronal membranes.

Several lines of evidence suggest that L-type Ca2+ channels (1,4-dihydropyridine receptors) are modulated by GTP-binding proteins. We have further examined this interaction by measuring the effect of 1,4-dihydropyridines on GTPase activity in brain membranes. Dihydropyridine agonists significantly increased GTPase, reflected by an increase in the maximal rate of GTP hydrolysis, without affecting the affinity for GTP or the binding of a non-hydrolysable analogue of GTP. The stimulating effect on GTPase was abolished by antisera raised against Go alpha but not Gi alpha. L-type Ca2+ channels may act as endogenous GTPase activating proteins (GAPs) to stimulate GTP hydrolysis by Go.

Modelling of a voltage-dependent Ca2+ channel beta subunit as a basis for understanding its functional properties.

Structure prediction methods have been used to establish a domain structure for the voltage-dependent calcium channel beta subunit, beta1b. One domain was identified from homology searches as an SH3 domain, whilst another was shown, using threading algorithms, to be similar to yeast guanylate kinase. This domain structure suggested relatedness to the membrane-associated guanylate kinase protein family, and that the N-terminal domain of the beta subunit might be similar to a PDZ domain. Three-dimensional model structures have been constructed for these three domains. The extents of the domains are consistent with functional properties and mutational assays of the subunit, and provide a basis for understanding its modulatory function.

Voltage-dependent calcium channel beta-subunits in combination with alpha 1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by G alpha o in rat frontal cortex.

The dihydropyridine-sensitive calcium channel agonist (-)-BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by Go in rat frontal cortex membranes. An anti-calcium channel beta-subunit antiserum abolished the (-)-BayK 8644-stimulated hydrolysis of GTP by Go and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the beta-subunit binding domain of the calcium channel complex, also attenuated (-)-BayK 8644 activation of GTPase. This study suggests that the calcium channel beta-subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Go. This may be a mechanism by which calcium channels can normally act to limit the duration of a G-protein modulatory signal.

Use of site-directed antibodies to probe the topography of the alpha 2 subunit of voltage-gated Ca2+ channels.

Polyclonal antibodies were raised against peptides corresponding to residues 1-15, 469-483 and 933-951 of the rabbit skeletal muscle L-type calcium channel alpha 2/delta primary translation product, for use as topological probes. Immunocytochemical comparison of the abilities of the antibodies to bind to the alpha 2 and delta subunits in intact and detergent-permeabilised rat dorsal root ganglion cells enabled the membrane orientation of these regions to be established. The resultant data indicate that the regions containing residues 1-15 and 469-483 of the alpha 2 subunit, and residues 1-17 of the delta subunit, are exposed on the extracellular surface of the membrane, findings consistent with a model that proposes alpha 2 to be entirely extracellular.

Direct and indirect modulation of neuronal calcium currents by G-protein activation.

Evidence is presented for modulation of the various components of neuronal Ca2+ currents by G-proteins and by gamma-aminobutyric acid B (GABAB) receptor activation. The mechanism of these interactions and the possible involvement of second messengers is discussed. The role of inhibition of Ca2+ channels in presynaptic inhibition of neurotransmitter release is examined.

CaVbeta subunit-mediated up-regulation of CaV2.2 currents triggered by D2 dopamine receptor activation.

Voltage-dependent Ca(2+) channels (VDCCs) are subject to modulation by a number of pathways, including membrane-delimited inhibition by heterotrimeric G-proteins and modulation through phosphorylation by diverse kinases. Here we report that in the Xenopus oocyte expression system Ca(V)2.2 channels undergo a sustained, linear and irreversible run-up lasting up to 30 min, which is potentiated during G-protein-mediated inhibition by activation of co-expressed G-protein coupled receptors (GPCRs). This up-regulation is not a result of receptor desensitization, but is associated with a hyperpolarization of the voltage for activation and depends on the presence of accessory subunits such that beta subunits promote, and alpha2delta subunits oppose the current increase. We have investigated the involvement of G-proteins and found that over-expression of Galpha(o) subunits or Galpha-transducin reduced the amount of agonist-mediated up-regulation. However, we have found no evidence for the involvement of any second messenger pathways in the increase of current run-up in the presence of a GPCR agonist. Taken together, our data suggest that the effect reported herein involves an enhancement of the GTPase activity of endogenous Galpha subunits, which is triggered by GPCR activation and mediated by accessory Ca(V)beta subunits. It may involve an increased association of Ca(V)beta subunits with alpha1 subunits in the plasma membrane or trafficking of channels to the plasma membrane.

Overlapping selectivity of neurotoxin and dihydropyridine calcium channel blockers in cerebellar granule neurones.

Calcium (Ca(2+)) currents have been studied extensively in cerebellar granule neurones, but much of the whole-cell pharmacology is inconsistent. Ca(2+) channel currents were recorded from granule neurones to investigate whether the commonly used Ca(2+) channel blockers show overlapping selectivity. Using combinations of toxin channel blockers, 45% of the total current was shown to be carried by Ca(2+) channels susceptible to block by the combined, or cumulative application of, omega-agatoxin IVA, omega-conotoxin GVIA and omega-conotoxin MVIIC, thus representing P/Q- and N-type channel currents. However, sequential application of these toxins showed that substantial overlap occurred in the proportions of current sensitive to individual toxins. Application of the 1, 4-dihydropyridine nicardipine at 1 microM, a concentration reported to be selective for L-type channels, blocked 16% of the total current, without reducing the current sensitive to the toxins used. However, greater concentrations of nicardipine (>10 microM) blocked a proportion of the total current that could not be accounted for by L-type channels alone. These results demonstrate that a pharmacological approach based on the L, N, P/Q, and R classification does not adequately describe the Ca(2+) channel subtypes found in cerebellar granule neurones due to substantial cross-selectivity to the drugs and toxins used.

Ca2+ currents in cerebellar granule neurones: role of internal Mg2+ in altering characteristics and antagonist effects.

Using the whole-cell patch-clamp technique, Ca2+ channel currents were measured in cultured rat cerebellar granule neurones in the presence of 10 mM Ba2+. Two different solutions were used to fill patch pipettes, one containing mainly tetraethylammonium acetate (TEA-Ac solution), and the other mainly caesium and HEPES (Cs-HEPES solution). Under these two different intracellular conditions markedly different Ca2+ channel currents were recorded. When TEA-Ac solution was used intracellularly, small, Cd(2+)-sensitive inward currents (approx. -55 pA) that were inhibited by the dihydropyridine antagonist (-)-202-791 and the GABAB agonist (-)-baclofen were observed. These currents were insensitive to the Ca2+ channel clocking toxins omega-conotoxin GVIA (omega-CgTX) and omega-agatoxin IVA and were enhanced by the dihydropyridine agonist (+)-202-791. In contrast, when the Cs-HEPES solution was used, currents were 2-3 times larger (approx. -130 pA), inhibited by (-)-202-791, omega-CgTX and omega-agatoxin IVA but were unaffected by (-)-baclofen. Furthermore, both (+)-202-791 and Bay K8644 in the presence of Cs-HEPES solution produced only a transient enhancement that was followed by an inhibition. Analysis of steady-state inactivation revealed two components of current in both cases, with similar voltage dependencies. The factor(s) giving rise to these differences were investigated in terms of current amplitude and responses to (-)-baclofen and omega-CgTX and were found to be mainly due to the concentrations of Mg2+ and ATP added to the patch pipette solutions. Furthermore, free internal Mg2+ concentrations of greater than 0.2 mM selectively inhibited omega-CgTX-sensitive Ca2+ channels. Preliminary evidence indicates that the same may be true of omega-Aga IVA-sensitive P-type current. These data suggest that the N-type Ca2+ channels in these cells are preferentially inhibited by intracellular Mg2+ and this may provide an explanation for discrepancies between the results of different groups investigating Ca2+ channel currents in similar cell types.

The excitatory amino-acid antagonist gamma-D-glutamylglycine masks rather than prevents long term potentiation of the perforant path.

The effect of the glutamate antagonist gamma-D-glutamylglycine on the induction of long-term potentiation in the dentate gyrus has been investigated in vivo. gamma-D-glutamyglycine (10(-3) M) perfused through a push-pull cannula into the dentate gyrus, rapidly reduced perforant path evoked potentials. Application to the perforant path of a high-frequency train (250 Hz, 500 ms), which in control animals reliably produced long term potentiation, had no effect on the evoked potentials when applied during blockade by gamma-D-glutamylglycine. This result was obtained despite the inability of gamma-D-glutamylglycine completely to inhibit the evoked potentials. However, when standard medium was reintroduced, potentiation was revealed in animals that had received the high-frequency train, whereas in animals that had received no high-frequency train during gamma-D-glutamylglycine inhibition the potentials returned only to pre-drug levels. In additional experiments, in which the dentate gyrus was continuously perfused with [3H]glutamine, and the steady state outflow of [3H]glutamate was measured, it was observed that gamma-D-glutamylglycine (10(-3) M) increased the steady state release of [3H]glutamate into the perfusate. From this result it is likely that gamma-D-glutamylglycine does not have any presynaptic inhibitory activity at the perforant path-granule cell synapse. The results indicate that a high frequency train applied to the perforant path during a period of inhibition by gamma-D-glutamylglycine was able to induce long term potentiation, whose expression was, however, masked until the glutamate antagonist was removed.(ABSTRACT TRUNCATED AT 250 WORDS)

G-protein mediation in nociceptive signal transduction: an investigation into the excitatory action of bradykinin in a subpopulation of cultured rat sensory neurons.

Bradykinin is one of several pro-inflammatory, pain-inducing substances produced during inflammation--the body's response to injury. In previous work we have shown that bradykinin and guanosine-5'-O-3-thiotriphosphate increase excitability in a subpopulation of cultured neonatal rat dorsal root ganglion neurons. We now describe experiments in which the mechanism underlying the stimulatory action of these two substances has been examined in more detail. Using the whole-cell voltage-clamp technique, bradykinin-sensitive cells were distinguished by their response to a 1-s depolarizing voltage-pulse which evoked more than one inward current during the step command. The secondary inward currents are likely to represent action potentials generated at the poorly clamped neurites of these cells. Bradykinin- and guanosine-5'-O-3-thiotriphosphate-induced changes in excitability were measured indirectly by a change in the number of inward currents recorded during the 1-s depolarizing voltage-step. The effect of activators and inhibitors of protein kinase C, arachidonic acid metabolism, G-protein activation and release of intracellular Ca2+ were examined on this response. In the presence of extracellular staurosporine (1.0 microM) or nordihydroguaiaretic acid (10 microM), these excitatory effects were reduced but not abolished, whilst indomethacin (20 microM) had no effect. Intracellular application of guanosine-5'-O-2-thiodiphosphate (10 mM) or ryanodine (100 microM) substantially reduced the effect of bradykinin. The excitatory effect of internal guanosine-5'-O-3-thiotriphosphate (500 microM) occurred gradually over time, and this was mimicked by internal application of myo-inositol 1,4,5-trisphosphorothioate (1.0 microM). From the results, it is proposed that G-protein activation is an essential component of the bradykinin response, which may also require a Ca(2+)-activated conductance modulated by protein kinase C and lipoxygenase metabolites of arachidonic acid.

A comparison of the effect of calcium channel ligands and GABAB agonists and antagonists on transmitter release and somatic calcium channel currents in cultured neurons.

Glutamate release has been examined from cultured cerebellar granule neurons in the rat using the technique of prelabelling the releasable pool of glutamate with [3H]glutamine. Glutamate release was stimulated in control neurons by 2-min incubation with 50 mM K+, or in neurons continuously depolarized in Ca2(+)-free 50 mM K+ medium, by 2-min incubation with medium containing 5 mM Ca2+. The ability of the Ca2(+)-channel agonist (+)-202-791 to increase the stimulated release of [3H]glutamate was approximately doubled in the depolarized condition. The antagonist enantiomer (-)-202-791 produced a small inhibition of K(+)-stimulated release, whereas (-)-202-791 completely inhibited Ca2(+)-stimulated release from depolarized neurons at concentrations greater than 10 nM. (-)-Baclofen (100 microM) inhibited transmitter release similarly (25-30%) under the two conditions. Calcium-channel currents were recorded from cultured dorsal root ganglion neurons under control conditions at a holding potential of -80 mV, or in neurons depolarized to -30 mV. (-)-202-791 produced a greater effect at -30 than at -80 mV although even at -30 mV the inhibition was slow in onset and incomplete. (-)-Baclofen (100 microM) inhibited the amplitude of the calcium-channel current at both holding potentials by 30-50%, although it did not clearly slow activation of the current at the depolarized holding potential. The GABAB receptors associated with inhibition of glutamate release and of calcium-channel currents were both markedly blocked by phaclofen but not by 2-OH-saclofen. These findings suggest that the GABAB receptor associated with inhibitory modulation of transmitter release, and that associated with inhibition of calcium-channel currents show pharmacological similarities, and are able to exert their action even at levels of steady depolarization at which most N-type channels should be inactivated.