Does modulation of the endocannabinoid system have potential therapeutic utility in cerebellar ataxia?

Cerebellar ataxias represent a spectrum of disorders which are, however, linked by common symptoms of motor incoordination and typically associated with deficiency in Purkinje cell firing activity and, often, degeneration. Cerebellar ataxias currently lack a curative agent. The endocannabinoid (eCB) system includes eCB compounds and their associated metabolic enzymes, together with cannabinoid receptors, predominantly the cannabinoid CB1 receptor (CB1 R) in the cerebellum; activation of this system in the cerebellar cortex is associated with deficits in motor coordination characteristic of ataxia, effects which can be prevented by CB1 R antagonists. Of further interest are various findings that CB1 R deficits may also induce a progressive ataxic phenotype. Together these studies suggest that motor coordination is reliant on maintaining the correct balance in eCB system signalling. Recent work also demonstrates deficient cannabinoid signalling in the mouse 'ducky(2J) ' model of ataxia. In light of these points, the potential mechanisms whereby cannabinoids may modulate the eCB system to ameliorate dysfunction associated with cerebellar ataxias are considered.

Cannabidivarin is anticonvulsant in mouse and rat.

BACKGROUND AND PURPOSE: Phytocannabinoids in Cannabis sativa have diverse pharmacological targets extending beyond cannabinoid receptors and several exert notable anticonvulsant effects. For the first time, we investigated the anticonvulsant profile of the phytocannabinoid cannabidivarin (CBDV) in vitro and in in vivo seizure models. EXPERIMENTAL APPROACH: The effect of CBDV (1-100 µM) on epileptiform local field potentials (LFPs) induced in rat hippocampal brain slices by 4-aminopyridine (4-AP) application or Mg(2+) -free conditions was assessed by in vitro multi-electrode array recordings. Additionally, the anticonvulsant profile of CBDV (50-200 mg·kg(-1) ) in vivo was investigated in four rodent seizure models: maximal electroshock (mES) and audiogenic seizures in mice, and pentylenetetrazole (PTZ) and pilocarpine-induced seizures in rats. The effects of CBDV in combination with commonly used antiepileptic drugs on rat seizures were investigated. Finally, the motor side effect profile of CBDV was investigated using static beam and grip strength assays. KEY RESULTS: CBDV significantly attenuated status epilepticus-like epileptiform LFPs induced by 4-AP and Mg(2+) -free conditions. CBDV had significant anticonvulsant effects on the mES (≥100 mg·kg(-1) ), audiogenic (≥50 mg·kg(-1) ) and PTZ-induced seizures (≥100 mg·kg(-1) ). CBDV (200 mg·kg(-1) ) alone had no effect against pilocarpine-induced seizures, but significantly attenuated these seizures when administered with valproate or phenobarbital at this dose. CBDV had no effect on motor function. CONCLUSIONS AND IMPLICATIONS: These results indicate that CBDV is an effective anticonvulsant in a broad range of seizure models. Also it did not significantly affect normal motor function and, therefore, merits further investigation as a novel anti-epileptic in chronic epilepsy models. LINKED ARTICLES: This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8.

Investigation of the effects of the novel anticonvulsant compound carisbamate (RWJ-333369) on rat piriform cortical neurones in vitro.

BACKGROUND AND PURPOSE: Carisbamate is being developed for adjuvant treatment of partial onset epilepsy. Carisbamate produces anticonvulsant effects in primary generalized, complex partial and absence-type seizure models, and exhibits neuroprotective and antiepileptogenic properties in rodent epilepsy models. Phase IIb clinical trials of carisbamate demonstrated efficacy against partial onset seizures; however, its mechanisms of action remain unknown. Here, we report the effects of carisbamate on membrane properties, evoked and spontaneous synaptic transmission and induced epileptiform discharges in layer II-III neurones in piriform cortical brain slices. EXPERIMENTAL APPROACH: Effects of carisbamate were investigated in rat piriform cortical neurones by using intracellular electrophysiological recordings. KEY RESULTS: Carisbamate (50-400 micromol x L(-1)) reversibly decreased amplitude, duration and rise-time of evoked action potentials and inhibited repetitive firing, consistent with use-dependent Na+ channel block; 150-400 micromol x L(-1) carisbamate reduced neuronal input resistance, without altering membrane potential. After microelectrode intracellular Cl(-) loading, carisbamate depolarized cells, an effect reversed by picrotoxin. Carisbamate (100-400 micromol x L(-1)) also selectively depressed lateral olfactory tract-afferent evoked excitatory synaptic transmission (opposed by picrotoxin), consistent with activation of a presynaptic Cl(-) conductance. Lidocaine (40-320 micromol x L(-1)) mimicked carisbamate, implying similar modes of action. Carisbamate (300-600 micromol x L(-1)) had no effect on spontaneous GABA(A) miniature inhibitory postsynaptic currents and at lower concentrations (50-200 micromol x L(-1)) inhibited Mg2+-free or 4-aminopyridine-induced seizure-like discharges. CONCLUSIONS AND IMPLICATIONS: Carisbamate blocked evoked action potentials use-dependently, consistent with a primary action on Na+ channels and increased Cl(-) conductances presynaptically and, under certain conditions, postsynaptically to selectively depress excitatory neurotransmission in piriform cortical layer Ia-afferent terminals.

Effects of Delta9-tetrahydrocannabivarin on [35S]GTPgammaS binding in mouse brain cerebellum and piriform cortex membranes.

BACKGROUND AND PURPOSE: We have recently shown that the phytocannabinoid Delta9-tetrahydrocannabivarin (Delta9-THCV) and the CB1 receptor antagonist AM251 increase inhibitory neurotransmission in mouse cerebellum and also exhibit anticonvulsant activity in a rat piriform cortical (PC) model of epilepsy. Possible mechanisms underlying cannabinoid actions in the CNS include CB1 receptor antagonism (by displacing endocannabinergic tone) or inverse agonism at constitutively active CB1 receptors. Here, we investigate the mode of cannabinoid action in [35S]GTPgammaS binding assays. EXPERIMENTAL APPROACH: Effects of Delta9-THCV and AM251 were tested either alone or against WIN55,212-2-induced increases in [35S]GTPgammaS binding in mouse cerebellar and PC membranes. Effects on non-CB receptor expressing CHO-D2 cell membranes were also investigated. KEY RESULTS: Delta9-THCV and AM251 both acted as potent antagonists of WIN55,212-2-induced increases in [35S]GTPgammaS binding in cerebellar and PC membranes (Delta9-THCV: pA2=7.62 and 7.44 respectively; AM251: pA2=9.93 and 9.88 respectively). At micromolar concentrations, Delta9-THCV or AM251 alone caused significant decreases in [35S]GTPgammaS binding; Delta9-THCV caused larger decreases than AM251. When applied alone in CHO-D2 membranes, Delta9-THCV and AM251 also caused concentration-related decreases in G protein activity. CONCLUSIONS AND IMPLICATIONS: Delta9-THCV and AM251 act as CB1 receptors antagonists in the cerebellum and PC, with AM251 being more potent than Delta9-THCV in both brain regions. Individually, Delta9-THCV or AM251 exhibited similar potency at CB1 receptors in the cerebellum and the PC. At micromolar concentrations, Delta9-THCV and AM251 caused a non-CB receptor-mediated depression of basal [35S]GTPgammaS binding.

The phytocannabinoid Delta(9)-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum.

BACKGROUND AND PURPOSE: The phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS. EXPERIMENTAL APPROACH: Effects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro. KEY RESULTS: The cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB(1) receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide. CONCLUSIONS AND IMPLICATIONS: We show for the first time that Delta(9)-THCV acts as a functional CB(1) receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB(1) receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV- and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.

Critical dynamics of gauge systems: spontaneous vortex formation in 2D superconductors.

We examine the formation of vortices during the nonequilibrium relaxation of a high-temperature initial state of an Abelian-Higgs system. We equilibrate the scalar and gauge fields using gauge-invariant Langevin equations and relax the system by instantaneously removing thermal fluctuations. For couplings near critical, kappa(c) = square root[lambda]/e = 1, we observe the formation of large clusters of like-sign magnetic vortices. Their appearance has implications for the dynamics of the phase transition, for the distribution of topological defects, and for late-time phase ordering kinetics. We offer explanations for both the observed vortex densities and vortex configurations.

The Cav2.1/alpha1A (P/Q-type) voltage-dependent calcium channel mediates inhibitory neurotransmission onto mouse cerebellar Purkinje cells.

The effects of voltage-dependent calcium channel (VDCC) antagonists on spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse Purkinje cells were examined using in vitro cerebellar slices. The inorganic ion Cd2+ reduced sIPSC amplitude and frequency. No additional block was seen with the Na+ channel antagonist tetrodotoxin (TTX) suggesting that all action potential-evoked inhibitory GABA release was mediated by high-voltage-activated VDCCs. No evidence was found for involvement of Cav1/alpha1C and alpha1D (L-type), Cav2.2/alpha1B (N-type) or Cav2.3/alpha1E (R-type) high-voltage-activated VDCCs or low-voltage-activated Cav3/alpha1G, alpha1H and alpha1I (T-type) VDCCs in mediating presynaptic GABA release. Blockade of sIPSCs by 200 nM omega-agatoxin IVA implicated the Cav2.1/alpha1A (P/Q-type) subtype of high-voltage-activated VDCCs in mediating inhibitory transmission. Inhibition by omega-agatoxin IVA was similar to that seen with Cd2+ and TTX. Selective antibodies directed against the Cav2.1 subunit revealed staining in regions closely opposed to Purkinje cell somata. Cav2.1 staining was colocalized with staining for antibodies against glutamic acid decarboxylase and corresponded well with the pericellular network formed by GABAergic basket cell interneurons. Antibody labelling of Cav2.3 revealed a region-specific expression. In the cerebellar cortex anterior lobe, Cav2.3 staining was predominantly somatodendritic; whilst in the posterior lobe, perisomatic staining was seen primarily. However, electrophysiological data was not consistent with a role for the Cav2.3 subunit in mediating presynaptic GABA release. No consistent staining was seen for other Cav (alpha1) subunits. Electrophysiological and immunostaining data support a predominant role for Cav2.1 subunits in mediating action potential-evoked inhibitory GABA release onto mouse Purkinje cells.

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma.

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.

Hyperpolarization-activated currents in presynaptic terminals of mouse cerebellar basket cells.

Using patch-clamp techniques, a hyperpolarization-activated current (I(h)) was recorded from synaptic terminals of mouse cerebellar basket cells. Ih was blocked quickly and reversibly by 2 mM Cs(+), and subtraction revealed a rapidly activating and deactivating I(h) current. Similar gating and block of presynaptic I(h) were also seen with the more selective inhibitor ZD 7288 (10 microM). The time constant of activation (tau (a))of presynaptic I(h) current became faster with membrane hyperpolarization, being approximately 74 ms at -130 mV, changing e-fold for a 33 mV change in membrane potential. Whole-cell recordings from basket cell somata also revealed an I(h) current, which was similarly sensitive to block by ZD 7288. Inhibition of I(h) by 10 microM ZD 7288 reduced the frequency ( approximately 34 %) and amplitude ( approximately 26 %) of spontaneous IPSCs (sIPSCs) recorded in Purkinje cells, one of the principal synaptic targets of basket neurones. This is the first report of an I(h) current in mammalian inhibitory presynaptic terminals, which may be an important target for neuromodulation in the cerebellum. Comparing the biophysical properties and distribution of cloned hyperpolarization-activated cation channels, we also suggest a molecular candidate underlying I(h) at these synapses.

The alpha1B Ca2+ channel amino terminus contributes determinants for beta subunit-mediated voltage-dependent inactivation properties.

Co-expression of auxiliary beta subunits with the alpha1B Ca2+ channel subunit in COS-7 cells resulted in an increase in current density and a hyperpolarising shift in the mid-point of activation. Amongst the beta subunits, beta2a in particular, but also beta4 and beta1b caused a significant retardation of the voltage-dependent inactivation compared to currents with alpha1B alone, whilst no significant changes in inactivation properties were seen for the beta3 subunit in this system. Prevention of beta2a palmitoylation, by introducing cysteine to serine mutations (beta2a(C3,4S)), greatly reduced the ability of beta2a to retard voltage-dependent inactivation. Deletion of the proximal half of the alpha1B cytoplasmic amino terminus (alpha1BDelta1-55) differentially affected beta subunit-mediated voltage-dependent inactivation properties. These effects were prominent with the beta2a subunit and, to a lesser extent, with beta1b. For beta2a, the major effects of this deletion were a partial reversal of beta2a-mediated retardation of inactivation and the introduction of a fast component of inactivation, not seen with full-length alpha1B. Deletion of the amino terminus had no other major effects on the measured biophysical properties of alpha1B when co-expressed with beta subunits. Transfer of the whole alpha1B amino terminus into alpha1C (alpha1bCCCC) conferred a similar retardation of inactivation on alpha1C when co-expressed with beta2a to that seen in parental alpha1B. Individual (alpha1B(Q47A) and alpha1B(R52A)) and double (alpha1B(R52,54A)) point mutations within the amino terminus of alpha1B also opposed the beta2a-mediated retardation of alpha1B inactivation kinetics. These results indicate that the alpha1B amino terminus contains determinants for beta subunit-mediated voltage-dependent inactivation properties. Furthermore, effects were beta subunit selective. As deletion of the alpha1B amino terminus only partially opposed beta subunit-mediated changes in inactivation properties, the amino terminus is likely to contribute to a complex site necessary for complete beta subunit function.

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.

Dissection of the calcium channel domains responsible for modulation of neuronal voltage-dependent calcium channels by G proteins.

The molecular determinants for G-protein regulation of neuronal calcium channels remain controversial. We have generated a series of alpha 1B/alpha 1E chimeric channels, since rat brain alpha 1E (rbEII), unlike human alpha 1E, showed no G-protein modulation. The study, carried out in parallel using D2 receptor modulation of calcium currents in Xenopus oocytes of G beta gamma modulation of calcium currents in COS-7 cells, consistently showed an essential role for domain I (from the N terminus to the end of the I-II loop) of the alpha 1B Ca2+ channel in G-protein regulation, with no additional effect of the C terminal of alpha 1B. The I-II loop alone of alpha 1B, or the I-II loop together with the C-terminal tail, was insufficient to confer G-protein modulation of alpha 1E (rbEII). We have further observed that the alpha 1E clone rbEII is truncated at the N-terminus compared to other alpha 1 subunits, and we isolated a PCR product from rat brain equivalent to a longer N-terminal isoform. The long N-terminal alpha 1E, unlike the short form, showed G-protein modulation. Furthermore, the equivalent truncation of alpha 1B (delta N1-55) abolished G-protein modulation of alpha 1B. Thus, we propose that the N terminus of alpha 1B and alpha 1E calcium channels contains essential molecular determinants for membrane-delimited G-protein inhibition, and that other regions, including the I-II loop and the C terminus, do not play a conclusive role alone.

Facilitation of rabbit alpha1B calcium channels: involvement of endogenous Gbetagamma subunits.

1. The alpha1B (N-type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gbetagamma subunits. Using transient expression in COS-7 cells of alpha1B together with the accessory subunits alpha2-delta and beta2a, we have examined the role of endogenous Gbetagamma subunits in the tonic modulation of alpha1B, and compared this with modulation by exogenously expressed Gbetagamma subunits. 2. Prepulse facilitation of control alpha1B/alpha2-delta/beta2a currents was always observed. This suggests the existence of tonic modulation of alpha1B subunits. To determine whether endogenous Gbetagamma is involved in the facilitation observed in control conditions, the betaARK1 Gbetagamma-binding domain (amino acids 495-689) was overexpressed, in order to bind free Gbetagamma subunits. The extent of control prepulse-induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPbetaS also reduced the control facilitation. 3. Co-expression of the Gbeta1gamma2 subunit, together with the alpha1B/alpha2-delta/beta2a calcium channel combination, resulted in a marked degree of depolarizing prepulse-reversible inhibition of the whole-cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid-point of the voltage dependence of activation. Activation of endogenous Gbetagamma subunits by dialysis with GTPgammaS produced a smaller degree of prepulse-reversible inhibition. 4. The rate of reinhibition of alpha1B currents by activated G protein, following a depolarizing prepulse, was much faster with Gbeta1gamma2 than for the decay of facilitation in control cells. Furthermore, betaARK1 (495-689) co-expression markedly slowed the control rate of reinhibition, suggesting that the kinetics of reinhibition depend on the concentration of free endogenous or exogenously expressed Gbetagamma in the cells. In contrast, the rate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined. 5. These findings indicate that, in this system, the voltage-dependent facilitation of alpha1B that is observed under control conditions occurs as a result of endogenous free Gbetagamma binding to alpha1B.

Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E 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.

Role of domain I of neuronal Ca2+ channel alpha1 subunits in G protein modulation.

1. We studied the G protein inhibition of heteromultimeric neuronal Ca2+ channels by constructing a series of chimeric channels between the strongly modulated alpha1B subunit and the alpha1E(rbEII) subunit, which showed no modulation. 2. In parallel studies, alpha1 subunit constructs were co-expressed together with the accessory Ca2+ channel alpha2-delta and beta2a subunits in mammalian (COS-7) cells and Xenopus oocytes. G protein inhibition of expressed Ca2+ channel currents was induced by co-transfection of Gbeta1 and Ggamma2 subunits in COS-7 cells or activation of co-expressed dopamine (D2) receptors by quinpirole (100 nM) in oocytes. 3. The data indicate that transfer of the alpha1B region containing the N-terminal, domain I and the I-II loop (i.e. the alpha1B1-483 sequence), conferred G protein modulation on alpha1E(rbEII), both in terms of a slowing of activation kinetics and a reduction in current amplitude. 4. In contrast, the data are not consistent with the I-II loop and/or the C-terminal forming a unique site for G protein modulation.

Functional expression of rat brain cloned alpha1E calcium channels in COS-7 cells.

The properties of the rat brain alpha1E Ca2+ channel subunit and its modulation by accessory rat brain alpha2-delta and beta1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether alpha1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca2+ channel and to examine its pharmacology in detail. alpha1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric alpha1E/alpha2-delta/beta1b channels were largely dictated by the presence of the beta1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. alpha1E/alpha2-delta/beta1b channels did not appear to be regulated by Ca2+-induced inactivation. alpha1E was shown to exhibit a unique pharmacological profile. omega-Agatoxin IVA blocked the current in a dose-dependent manner with an IC50 of approximately 50 nM and a maximum inhibition of about 80%, whilst omega-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 micro;M) produced an inhibition of 51 +/- 7%, whereas the DHP agonist S-(-)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the alpha1E Ca2+ channel subunit; we propose that rat brain alpha1E forms a novel Ca2+ channel with properties more similar to a subtype of LVA than HVA Ca2+ current.

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.

Studies on the blocking action of human Kv3.4 inactivation peptide variants in the mouse cloned Kv1.1 K+ channel.

1. Whole-cell patch clamp recordings were made from Chinese hamster ovary (CHO) cells stably expressing homomeric mouse Kv1.1 (delayed rectifier K+; mKv1.1) channels. The effects of internal application of a number of different peptides, based on part of the amino terminal sequence of the human Kv3.4 channel subunit (hKv3.4), were examined in order to determine their influence on N-type inactivation. 2. For the native hKv3.4 peptide, the association rate constant (kon) increased with membrane depolarization, whilst the dissociation rate constant (koff) had little dependence on voltage. This resulted in the apparent dissociation constant (KD) of the hKv3.4 peptide tending to increase with depolarization. 3. In general, kon increased and apparent KD decreased with positive charge of the hKv3.4 peptide variants; in peptides lacking a hydrophobic amino terminal this correlation was not maintained. In contrast, the rate of dissociation of the variant peptides (koff) was independent of net charge. 4. The blocking activity of the hKv3.4 peptide was not dependent on a disulphide bridge between cysteine residues C6 and C24 and the presence of cysteine residues in the hKv3.4 peptide was not a prerequisite for rapid inactivation. All cysteine-substituted variants, especially at C6, showed a more rapid recovery from inactivation than the hKv3.4 peptide. Substitutions at C24, and not C6, reduced kon. 5. The present results concerning the action of the mammalian hKv3.4 channel inactivation particle on mKv1.1 channels complement earlier models for the invertebrate Shaker K+ channel. It is proposed that the hydrophobic amino terminal region of the hKv3.4 inactivation peptide blocks the channel pore, whilst the adjacent positively charged region interacts with negative charges on the channel protein.

The modulation of the rate of inactivation of the mKv1.1 K+ channel by the beta subunit, Kv beta 1 and lack of effect of a Kv beta 1 N-terminal peptide.

The coexpression of the rat Kv beta 1 subunit with the mouse Kv1.1 (mKv1.1) K+ channel in Chinese hamster ovary cells caused an increase in the rate of inactivation of whole-cell current. Current decayed in a bi-exponential fashion with a fast voltage-dependent and a slower voltage-independent component. The inactivating current component accounted for around 40% of the total outward current. In contrast to previous studies using K+ channel alpha subunits, peptides based on the N-terminal of the Kv beta 1 subunit were unable to mimic the action of the entire subunit. The findings indicate differences between the inactivation induced by the Kv beta 1 subunit and the N-type inactivation mechanism associated with certain rapidly-inactivating cloned K+ channel alpha subunits.

Cysteine-modifying reagents alter the gating of the rat cloned potassium channel Kv1.4.

The effects of cysteine-modifying reagents on the gating of rat cloned Kv1.4 channels expressed in HEK-293 cells were examined using the whole-cell patch-clamp technique. Cells transfected with Kv1.4 expressed a rapidly inactivating K+ current with a mid-point of activation of -31 mV and a slope factor of 5 mV measured with tail current protocols in 35 mM Rb+ external solutions. The cysteine-specific oxidizing agents 2,2'-dithiobis-5-nitropyridine (DTBNP, 50 microM) and chloramine-T (CL-T, 500 microM) removed inactivation of Kv1.4. These effects were reversed by the reducing agent dithiothreitol (DTT, 10mM). In addition, DTBNP and CL-T also slowed Kv1.4 deactivation and increased the voltage sensitivity of deactivation. The action of cysteine-modifying reagents on Kv1.4 suggests that redox state affects channel gating, with oxidation tending to stabilize the open state of the channel, both by removing inactivation and slowing deactivation.