Relief of G-protein inhibition of calcium channels and short-term synaptic facilitation in cultured hippocampal neurons.
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G-protein inhibition of voltage-gated calcium channels can be transiently relieved by repetitive physiological stimuli. Here, we provide evidence that such relief of inhibition contributes to short-term synaptic plasticity in microisland-cultured hippocampal neurons. With G-protein inhibition induced by the GABA(B) receptor agonist baclofen or the adenosine A1 receptor agonist 2-chloroadenosine, short-term synaptic facilitation emerged during action potential trains. The facilitation decayed with a time constant of approximately 100 msec. However, addition of the calcium channel inhibitor Cd(2+) at 2-3 microM had no such effect and did not alter baseline synaptic depression. As expected of facilitation from relief of channel inhibition, analysis of miniature EPSCs implicated presynaptic modulation, and elevating presynaptic Ca(2+) entry blunted the facilitation. Most telling was the near occlusion of synaptic facilitation after selective blockade of P/Q- but not N-type calcium channels. This was as predicted from experiments using recombinant calcium channels expressed in human embryonic kidney (HEK) 293 cells; we found significantly stronger relief of G-protein inhibition in recombinant P/Q- versus N-type channels during action potential trains. G-protein inhibition in HEK 293 cells was induced via recombinant M2 muscarinic acetylcholine receptors activated by carbachol, an acetylcholine analog. Thus, relief of G-protein inhibition appears to produce a novel form of short-term synaptic facilitation in cultured neurons. Similar short-term synaptic plasticity may be present at a wide variety of synapses, as it could occur during autoreceptor inhibition by glutamate or GABA, heterosynaptic inhibition by GABA, tonic adenosine inhibition, and in many other instances. (+info)
Combinatorial synthesis of omega-conotoxin MVIIC analogues and their binding with N- and P/Q-type calcium channels.
(10/164)
Omega-conotoxin MVIIC (MVIIC) blocks P/Q-type calcium channels with high affinity and N-type calcium channels with low affinity, while the highly homologous omega-conotoxin MVIIA blocks only N-type calcium channels. We wished to obtain MVIIC analogues more selective for P/Q-type calcium channels than MVIIC to elucidate structural differences among the channels, which discriminate the omega-conotoxins. To prepare a number of MVIIC analogues efficiently, we developed a combinatorial method which includes a random air oxidation step. Forty-seven analogues were prepared in six runs and some of them exhibited higher selectivity for P/Q-type calcium channels than MVIIC in binding assays. (+info)
Differential occurrence of reluctant openings in G-protein-inhibited N- and P/Q-type calcium channels.
(11/164)
Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel "slope" analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the "willing-reluctant" model, in which binding of G-protein betagamma subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca(2+) entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission. (+info)
Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.
(12/164)
Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis. (+info)
Abnormal transmitter release at neuromuscular junctions of mice carrying the tottering alpha(1A) Ca(2+) channel mutation.
(13/164)
Neurotransmitter release at many synapses is regulated by P/Q-type Ca(2+) channels containing the alpha(1A) pore-forming subunit. Mutations in alpha(1A) cause cerebral disorders including familial hemiplegic migraine (FHM) and ataxia in humans. Tottering (tg) alpha(1A) mutant mice display ataxia and epilepsy. It is not known whether alpha(1A) mutations induce impairment of synaptic function, which could underlie the symptoms of these cerebral disorders. To assess whether alpha(1A) mutations influence neurotransmitter release, we studied P-type Ca(2+) channel-mediated acetylcholine (ACh) release at tg neuromuscular junctions (NMJs) with micro-electrode measurements of synaptic potentials. We found a Ca(2+)-, Mg(2+)- and K(+)-dependent increase of spontaneous ACh release at both homo- and heterozygote tg NMJs. Furthermore, there was increased run-down of high-rate evoked release at homozygous tg NMJs. In isotonic contraction experiments this led to block of synaptic transmission at lower concentrations of the ACh antagonist tubocurarine than were needed in wild-type muscles. Our results suggest that in tg motor nerve terminals there is increased influx of Ca(2+) under resting conditions. This study shows that functional consequences of alpha(1A) mutations causing cerebral disorders can be characterized at the NMJ. (+info)
Binding of Ala-scanning analogs of omega-conotoxin MVIIC to N- and P/Q-type calcium channels.
(14/164)
omega-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P(2) membranes suggested that Thr(11), Tyr(13) and Lys(2) are essential for binding to both N- and P/Q-type channels, whereas Lys(4) and Arg(22) are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs. (+info)
Analysis of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene for expression in pancreatic islet beta cells using transgenic mice and HIT-T15 cells.
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The omega-agatoxin-IVA-sensitive P/Q-type Ca(2+) channel plays a role in insulin release from the pancreatic islets of beta cells. To dissect the molecular mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene using transgenic mice and HIT insulinoma cells. The E. coli lacZ reporter gene was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0. 5 kb of the 5'-upstream region failed to show reporter expression on histological examination. As the expression of alpha(1A)subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression. When linked to the placental alkaline phosphatase reporter gene to examine promoter activity for beta cell expression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but not the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells. The sequence between 3.0 and 1.5 kb upstream of the start codon enhanced thymidine kinase promoter activity in HIT cells, but not in fibroblast NIH3T3 cells. These results suggested that the beta cell-specific elements of the alpha(1A) subunit gene are likely to be located in the distal upstream region (-3021 to-1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negative cis-regulatory element(s) to suppress the alpha(1A) subunit gene expression in acini. (+info)
Abnormal CAG repeat expansion in the alpha1A voltage-dependent calcium channel gene is associated with spinocerebellar ataxia type 6, an autosomal dominant cerebellar ataxia with a predominant loss of the Purkinje cell. A reverse transcriptase-polymerase chain reaction analysis of mRNA from mouse Purkinje cells revealed a predominant expression of the alpha1A channel lacking an asparagine-proline (NP) stretch in the domain IV (alpha1A(-NP)). Human alpha1A channels carrying various polyglutamine length with or without NP were expressed in HEK293 cells, and channel properties were compared using a whole-cell voltage clamp technique. alpha1A(-NP), corresponding to P-type channel, with 24 and 28 polyglutamines found in patients showed the voltage dependence of inactivation shifting negatively by 6 and 11 mV, respectively, from the 13 polyglutamine control. Contrarily, the alpha1A channel with NP (alpha1A(+NP)), corresponding to Q-type channel, with 28 polyglutamines exhibited a positive shift of 5 mV. These results suggest that altered function of alpha1A(-NP) may contribute to degeneration of Purkinje cells, which express predominantly alpha1A(-NP), due to the reduced Ca(2+) influx resulting from the negative shift of voltage-dependent inactivation. On the other hand, other types of neurons, expressing both alpha1A(-NP) and alpha1A(+NP), may survive because the positive shift of voltage-dependent inactivation of alpha1A(+NP) compensates Ca(2+) influx. (+info)