Permissive effect of voltage on mGlu 7 receptor subtype signaling in neurons.
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G protein-coupled receptors mobilize neuronal signaling cascades which until now have not been shown to depend on the state of membrane depolarization. Thus we have previously shown that the metabotropic glutamate receptor type 7 (mGlu7 receptor) blocks P/Q-type Ca(2+) channels via activation of a G(o) protein and PKC, in cerebellar granule cells. We show here that the transient depolarizations used to evoke the studied Ca(2+) current were indeed permissive to activate this pathway by a mGlu7 receptor agonist. Indeed, sustained depolarization to 0 mV was sufficient to inhibit P/Q-type Ca(2+) channels. This effect involved a conformational change in voltage-gated sodium channel independently of Na(+) flux, activation of a pertussis toxin-sensitive G-protein, inositol trisphosphate formation, intracellular Ca(2+) release, and PKC activity. Subliminal sustained membrane depolarization became efficient in inducing inositol trisphosphate formation, release of intracellular Ca(2+) and in blocking Ca(2+) channels, when applied concomitantly with the mGlu7a receptor agonist, d,l-aminophosphonobutyrate. This synergistic effect of membrane depolarization and mGlu7 receptor activation provides a mechanism by which neuronal excitation could control action of the mGlu7 receptor in neurons. (+info)
Increased expression of alpha 1A Ca2+ channel currents arising from expanded trinucleotide repeats in spinocerebellar ataxia type 6.
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The expansion of polyglutamine tracts encoded by CAG trinucleotide repeats is a common mutational mechanism in inherited neurodegenerative diseases. Spinocerebellar ataxia type 6 (SCA6), an autosomal dominant, progressive disease, arises from trinucleotide repeat expansions present in the coding region of CACNA1A (chromosome 19p13). This gene encodes alpha(1A), the principal subunit of P/Q-type Ca(2+) channels, which are abundant in the CNS, particularly in cerebellar Purkinje and granule neurons. We assayed ion channel function by introduction of human alpha(1A) cDNAs in human embryonic kidney 293 cells that stably coexpressed beta(1) and alpha(2)delta subunits. Immunocytochemical analysis showed a rise in intracellular and surface expression of alpha(1A) protein when CAG repeat lengths reached or exceeded the pathogenic range for SCA6. This gain at the protein level was not a consequence of changes in RNA stability, as indicated by Northern blot analysis. The electrophysiological behavior of alpha(1A) subunits containing expanded (EXP) numbers of CAG repeats (23, 27, and 72) was compared against that of wild-type subunits (WT) (4 and 11 repeats) using standard whole-cell patch-clamp recording conditions. The EXP alpha(1A) subunits yielded functional ion channels that supported inward Ca(2+) channel currents, with a sharp increase in P/Q Ca(2+) channel current density relative to WT. Our results showed that Ca(2+) channels from SCA6 patients display near-normal biophysical properties but increased current density attributable to elevated protein expression at the cell surface. (+info)
The role of Kv1.2-containing potassium channels in serotonin-induced glutamate release from thalamocortical terminals in rat frontal cortex.
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Serotonin 5-HT(2A) receptors have been implicated in psychiatric illness and the psychotomimetic effects of hallucinogens. In brain slices, focal stimulation of 5-HT(2A) receptors in rat prefrontal cortex results in dramatically increased glutamate release onto layer V pyramidal neurons, as measured by an increase in "spontaneous" (nonelectrically evoked) EPSCs. This glutamate release is blocked by tetrodotoxin (TTX) and is thought to involve local spiking in thalamocortical axon terminals; however, the detailed mechanism has remained unclear. Here, we investigate parallels in EPSCs induced by either serotonin or the potassium channel blockers 4-aminopyridine (4-AP) or alpha-dendrotoxin (DTX). DTX, a selective blocker of Kv1.1-, Kv1.2-, and Kv1.6-containing potassium channels, has been shown to release glutamate in cortical synaptosomes, presumably by inhibiting a subthreshold-activated, slowly inactivating potassium conductance. By comparing DTX with other potassium channel blockers, we found that the ability to induce EPSCs in cortical pyramidal neurons depends on affinity for Kv1.2 subunits. DTX-induced EPSCs are similar to 5-HT-induced EPSCs in terms of sensitivity to TTX and omega-agatoxin-IVA (a blocker of P-type calcium channels) and laminar selectivity. The involvement of thalamocortical terminals in DTX-induced EPSCs was confirmed by suppression of these EPSCs by micro-opiates and thalamic lesions. More directly, DTX-induced EPSCs substantially occlude those induced by 5-HT, suggesting a common mechanism of action. No occlusion by DTX was seen when EPSCs were induced by a nicotinic mechanism. These results indicate that blockade of Kv1.2-containing potassium channels is part of the mechanism underlying 5-HT-induced glutamate release from thalamocortical terminals. (+info)
Dopamine modulates exocytosis independent of Ca(2+) entry in melanotropic cells.
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Dopamine is a known inhibitor of pituitary melanotropic cells. It reduces Ca(2+) influx by hyperpolarizing the cell membrane and by modulating high- and low-voltage-activated (HVA and LVA) Ca(2+) channels. As a result, dopamine reduces the hormonal output of the cell. However, it is unknown how dopamine affects each of the four different HVA Ca(2+) channel types individually. Moreover, it is unknown whether dopamine interacts with exocytosis independent of Ca(2+) channels. Here we show that dopamine differentially modulates the HVA Ca(2+) channels and that it affects the stimulus-secretion coupling through a direct effect on the exocytotic machinery. Sustained L- and P-type Ba(2+) currents are reduced in amplitude and inactivating N- and Q-type currents acquire different activation and inactivation kinetics in the presence of dopamine. The Q-type current shows slow activation, which is a hallmark for direct G-protein modulation. We used membrane capacitance measurements to monitor exocytosis. Surprisingly, we find that the amount of exocytosis per step depolarization is not diminished by dopamine despite the reduction in Ca(2+) current. To test whether dopamine affects the release machinery downstream of Ca(2+) entry, we stimulated exocytosis by dialyzing cells with buffered high-Ca(2+) solutions. Dopamine increased the amount and the rate of exocytosis. In the first 90 s, the rate of secretion was increased two- to threefold, but it was normalized again at 180 s, suggesting that predominantly vesicles that fuse early in the exocytotic phase are modulated by dopamine. Thus while Ca(2+) channels are inhibited by dopamine, the exocytotic machinery downstream of Ca(2+) influx is sensitized. As a result, release is more effectively stimulated by Ca(2+) influx during dopamine inhibition. (+info)
P/Q-type calcium-channel blockade in the periaqueductal gray facilitates trigeminal nociception: a functional genetic link for migraine?
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The discovery of mis-sense mutations in the alpha1A subunit of the P/Q-type calcium channel in patients with familial hemiplegic migraine indicates the potential involvement of dysfunctional ion channels in migraine. The periaqueductal gray (PAG) region of the brainstem modulates craniovascular nociception and, through its role in the descending pain modulation system, may contribute to migraine pathophysiology. In this study we sought to investigate the possible link between the genetic mutations found in migraineurs and the PAG as a modulator of craniovascular nociception. We microinjected the P/Q-type calcium-channel blocker omega-agatoxin IVA into the rat ventrolateral PAG (vlPAG). We examined its effect on the nociceptive transmission of second-order neurons recorded in the trigeminal nucleus caudalis and activated by stimulation of the parietal dura mater. After injection of agatoxin into the vlPAG (n = 20) responses to dural stimulation were facilitated by 143% (p < 0.0001) for Adelta-fiber activity and 180% for C-fiber activity (p < 0.05). Similarly, spontaneous background activity increased by 163% (p < 0.0001). These results demonstrate that P/Q-type calcium channels in the PAG play a role in modulating trigeminal nociception and suggest a role for dysfunctional P/Q-type calcium channels in migraine pathophysiology. (+info)
Differential facilitation of N- and P/Q-type calcium channels during trains of action potential-like waveforms.
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Inhibition of presynaptic voltage-gated calcium channels by direct G-protein betagamma subunit binding is a widespread mechanism that regulates neurotransmitter release. Voltage-dependent relief of this inhibition (facilitation), most likely to be due to dissociation of the G-protein from the channel, may occur during bursts of action potentials. In this paper we compare the facilitation of N- and P/Q-type Ca(2+) channels during short trains of action potential-like waveforms (APWs) using both native channels in adrenal chromaffin cells and heterologously expressed channels in tsA201 cells. While both N- and P/Q-type Ca(2+) channels exhibit facilitation that is dependent on the frequency of the APW train, there are important quantitative differences. Approximately 20 % of the voltage-dependent inhibition of N-type I(Ca) was reversed during a train while greater than 40 % of the inhibition of P/Q-type I(Ca) was relieved. Changing the duration or amplitude of the APW dramatically affected the facilitation of N-type channels but had little effect on the facilitation of P/Q-type channels. Since the ratio of N-type to P/Q-type Ca(2+) channels varies widely between synapses, differential facilitation may contribute to the fine tuning of synaptic transmission, thereby increasing the computational repertoire of neurons. (+info)
Kurtoxin, a gating modifier of neuronal high- and low-threshold ca channels.
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Studies of Ca channels expressed in oocytes have identified kurtoxin as a promising tool for functional and structural studies of low-threshold T-type Ca channels. This peptide, isolated from the venomous scorpion Parabuthus transvaalicus, inhibits low-threshold alpha1G and alpha1H Ca channels expressed in oocytes with relatively high potency and high selectivity. Here we report its effects on Ca channel currents, carried by 5 mm Ba(2+) ions, in rat central and peripheral neurons. In thalamic neurons 500 nm kurtoxin inhibited T-type Ca channel currents almost completely (90.2 +/- 2.5% at -85 mV; n = 6). Its selectivity, however, was less than expected because it also reduced the composite high-threshold Ca channel current recorded in these cells (46.1 +/- 6.9% at -30 mV; n = 6). In sympathetic and thalamic neurons, 250-500 nm kurtoxin partially inhibited N-type and L-type Ca channel currents, respectively. It similarly reduced the high-threshold Ca channel current that remains after a blockade of P-type, N-type, and L-type Ca channels in thalamic neurons. In contrast, kurtoxin facilitated steady-state P-type Ba currents in Purkinje neurons (by 34.9 +/- 3.7%; n = 10). In all cases the kurtoxin effect was voltage-dependent and entailed a modification of channel gating. Exposure to kurtoxin slowed current activation kinetics, although its effects on deactivation varied with the channel types. Kurtoxin thus appears as a unique gating-modifier that interacts with different Ca channel types with high affinity. This unusual property and the complex gating modifications it induces may facilitate future studies of gating in voltage-dependent ion channels. (+info)
Cdk5/p35 regulates neurotransmitter release through phosphorylation and downregulation of P/Q-type voltage-dependent calcium channel activity.
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Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase with close structural homology to the mitotic Cdks. The complex of Cdk5 and p35, the neuron-specific regulatory subunit of Cdk5, plays important roles in brain development, such as neuronal migration and neurite outgrowth. Moreover, Cdk5 is thought to be involved in the promotion of neurodegeneration in Alzheimer's disease. Cdk5 is abundant in mature neurons; however, its physiological functions in the adult brain are unknown. Here we show that Cdk5/p35 regulates neurotransmitter release in the presynaptic terminal. Both Cdk5 and p35 were abundant in the synaptosomes. Roscovitine, a specific inhibitor of Cdk5 in neurons, induced neurotransmitter release from the synaptosomes in response to membrane depolarization and enhanced the EPSP slopes in rat hippocampal slices. The electrophysiological study using each specific inhibitor of the voltage-dependent calcium channels (VDCCs) and calcium imaging revealed that roscovitine enhanced Ca2+ influx from the P/Q-type VDCC. Moreover, Cdk5/p25 phosphorylated the intracellular loop connecting domains II and III (L(II-III)) between amino acid residues 724 and 981 of isoforms cloned from rat brain of the alpha1A subunit of P/Q-type Ca2+ channels. The phosphorylation inhibited the interaction of L(II-III) with SNAP-25 and synaptotagmin I, which were plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins and were required for efficient neurotransmitter release. These results strongly suggest that Cdk5/p35 inhibits neurotransmitter release through the phosphorylation of P/Q-type VDCC and downregulation of the channel activity. (+info)