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Cav2.1 Channels (cav2.1 + channel)
Selected AbstractsThe carboxy-terminal tail region of human Cav2.1 (P/Q-type) channel is not an essential determinant for its subcellular localization in cultured neuronesGENES TO CELLS, Issue 2 2005Qiuping Hu A recent report on the mechanism of synaptic targeting of Cav2.2 channel suggested that this process depends upon the presence of long C-terminal tail and that protein interactions mediated by SH3-binding and PDZ-binding motifs in the tail region are important. To examine the possibility that C-terminal tail of the Cav2.1 channel and the polyglutamine stretch therein are also involved in the mechanism for channel localization, we constructed several expression plasmids for human Cav2.1 channel tagged with enhanced green fluorescent protein (EGFP) and introduced them into mouse hippocampal neuronal culture. HC construct encodes short version of Cav2.1, and HS and HL encode Cav2.1 channel with a long C-terminal tail, which contains polyglutamine tract of 13 (normal range) and 28 (SCA6 disease range) repeat units, respectively. Surprisingly, transfection with HC, HS, and HL gave essentially the same results: EGFP signal was observed in cell soma, dendrites, and the axon as well. Furthermore, mutation of the PDZ-binding motif located at the C-terminus of the long version of Cav2.1, by adding FLAG tag, did not affect the localization patterns of HS and HL as well. Therefore, the C-terminal region is not indispensable for the subcellular localization of Cav2.1 channel, nor expansion of polyglutamine length affected the localization of the channel. Thus, it is possible that the localization mechanism of Cav2.1 channel is different from that of Cav2.2, though these channels share various structural and functional characteristics. [source] Severely impaired neuromuscular synaptic transmission causes muscle weakness in the Cacna1a -mutant mouse rolling NagoyaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2007Simon Kaja Abstract The ataxic mouse rolling Nagoya (RN) carries a missense mutation in the Cacna1a gene, encoding the pore-forming subunit of neuronal Cav2.1 (P/Q-type) Ca2+ channels. Besides being the predominant type of Cav channel in the cerebellum, Cav2.1 channels mediate acetylcholine (ACh) release at the peripheral neuromuscular junction (NMJ). Therefore, Cav2.1 dysfunction induced by the RN mutation may disturb ACh release at the NMJ. The dysfunction may resemble the situation in Lambert,Eaton myasthenic syndrome (LEMS), in which autoantibodies target Cav2.1 channels at NMJs, inducing severely reduced ACh release and resulting in muscle weakness. We tested neuromuscular function of RN mice and characterized transmitter release properties at their NMJs in diaphragm, soleus and flexor digitorum brevis muscles. Clinical muscle weakness and fatigue were demonstrated using repetitive nerve-stimulation electromyography, grip strength testing and an inverted grid hanging test. Muscle contraction experiments showed a compromised safety factor of neuromuscular transmission. In ex vivo electrophysiological experiments we found severely impaired ACh release. Compared to wild-type, RN NMJs had 50,75% lower nerve stimulation-evoked transmitter release, explaining the observed muscle weakness. Surprisingly, the reduction in evoked release was accompanied by an ,,3-fold increase in spontaneous ACh release. This synaptic phenotype suggests a complex effect of the RN mutation on different functional Cav2.1 channel parameters, presumably with a positive shift in activation potential as a prevailing feature. Taken together, our studies indicate that the gait abnormality of RN mice is due to a combination of ataxia and muscle weakness and that RN models aspects of the NMJ dysfunction in LEMS. [source] The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006G. T. Macleod Abstract Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cacts2 mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cacts2 mutation occurs in the cytoplasmic carboxyl tail of the ,1 -subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+ -imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cacts2 mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Cav2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 °C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Cav2.1 channel inactivation. [source] Localization of synaptic proteins involved in neurosecretion in different membrane microdomainsJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Elena Taverna Abstract A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Cav2.1 (P/Q-type) channels and soluble N -ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Cav2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-,-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery. [source] | ||||||||||