Single calcium channels on a cholinergic presynaptic nerve terminal.

The calyx-type synapse of the chick ciliary ganglion was used to examine single calcium channels in a vertebrate cholinergic presynaptic nerve terminal by means of the cell-attached, patch-clamp technique. Calcium channels were recorded on the internal, transmitter-release face of the nerve terminal, but were not detected on the external face. These channels were recruited at -30 mV, with maximum activation at about +30 mV, and were sometimes clustered at high densities. Single-channel conductance estimates with voltage-pulse or -ramp techniques gave values of 11-14 pS with 110 mM barium, which is in the intermediate, N-type range for calcium channels on a control neuron. This nerve terminal calcium channel, termed the NPT-type, may link action potentials to transmitter release at many vertebrate fast-transmitting synapses.     

Co-expression of metabotropic glutamate receptor 7 and N-type Ca(2+) channels in single cerebrocortical nerve terminals of adult rats

The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine-tuning of neurotransmitter release. Here we report that, in cerebrocortical nerve terminals of adult rats, the inhibition of glutamate release is mediated by mGluR7. In this preparation, the major component of glutamate release is supported by P/Q-type Ca2+ channels (72.7%). However, mGluR7 selectively reduced the release component that is associated with N-type Ca2+ channels (29.9%). Inhibition of P/Q channels by mGluR7 is not masked by the higher efficiency of these channels in driving glutamate release when compared with N-type channels. Thus, activation of mGluR7 failed to reduce the release associated with P/Q channels when the extracellular calcium concentration, ([Ca2+]o), was reduced from 1.3 to 0.5 mm. Through Ca2+ imaging, we show that Ca2+ channels are distributed in a heterogeneous manner in individual nerve terminals. Indeed, in this preparation, nerve terminals were observed that contain N-type (31.1%; conotoxin GVIA-sensitive) or P/Q-type (64.3%; agatoxin IVA-sensitive) channels or that were insensitive to these two toxins (4.6%). Interestingly, the great majority of the responses to l-AP4 (95.4%) were observed in nerve terminals containing N-type channels. This specific co-localization of mGluR7 and N-type Ca2+-channels could explain the failure of the receptor to inhibit the P/Q channel-associated release component and also reveal the existence of specific targeting mechanisms to localize the two proteins in the same nerve terminal subset.     

Molecular cloning and characterization of a novel calcium channel from rabbit brain.

The complete amino acid sequence of a novel calcium channel (designated BII) from rabbit brain has been deduced by cloning and sequencing the cDNA. The BII calcium channel is structurally more closely related to the BI calcium channel than to the cardiac and skeletal muscle L-type calcium channels. Blot hybridization analysis of RNA from different tissues and from different regions of the brain shows that the BII calcium channel is distributed predominantly in the brain, being abundant in the cerebral cortex, hippocampus and corpus striatum.     

The neurobiology of antiepileptic drugs for the treatment of nonepileptic conditions

Antiepileptic drugs (AEDs) are commonly prescribed for nonepileptic conditions, including migraine headache, chronic neuropathic pain, mood disorders, schizophrenia and various neuromuscular syndromes. In many of these conditions, as in epilepsy, the drugs act by modifying the excitability of nerve (or muscle) through effects on voltage-gated sodium and calcium channels or by promoting inhibition mediated by gamma-aminobutyric acid (GABA) A receptors. In neuropathic pain, chronic nerve injury is associated with the redistribution and altered subunit compositions of sodium and calcium channels that predispose neurons in sensory pathways to fire spontaneously or at inappropriately high frequencies, often from ectopic sites. AEDs may counteract this abnormal activity by selectively affecting pain-specific firing; for example, many AEDs suppress high-frequency action potentials by blocking voltage-activated sodium channels in a use-dependent fashion. Alternatively, AEDs may specifically target pathological channels; for example, gabapentin is a ligand of alpha2delta voltage-activated calcium channel subunits that are overexpressed in sensory neurons after nerve injury. Emerging evidence suggests that effects on signaling pathways that regulate neuronal plasticity and survival may be a factor in the delayed clinical efficacy of AEDs in some neuropsychiatric conditions, including bipolar affective disorder.     

Voltage-dependent P/Q-type calcium channels at the frog neuromuscular junction

It is well known that antagonists of N-type voltage-gated calcium channels inhibit the evoked quantal release of acetylcholine in amphibian neuromuscular synapses. This, however, does not exclude the functional expression of other types of voltage-gated calcium channels in these nerve terminals. Using immunocytochemistry, we detected the expression of the alpha1A subunit of P/Q-type calcium channels (that is otherwise typical of mammalian motor nerve endings) in the frog neuromuscular junction. In addition, we demonstrated that the P/Q-type channel blocker omega-agatoxin IVA (20 nM) reduced the action potential-induced calcium transient and significantly decreased both spontaneous and evoked mediator release. Our data indicates the functional expression of P/Q-type calcium channels in the frog motor nerve ending which participate in acetylcholine release.     

cDNA cloning and characterization of an osmotically sensitive TRP channel from ascidian eggs

The transient receptor potential (TRP) ion channels are thought to be involved in the entry of calcium ion into cells. In this study, we isolated a cDNA clone, HrTRPV, that shows high homology to Caenorhabditis elegans OSM-9, a TRPV subfamily member of the TRP family, from a Halocynthia roretzi fertilized egg cDNA library. We analyzed its properties using HrTRPV-transfected cells. Upon reduction of extracellular osmolarity, the intracellular calcium concentration was found to increase in HrTRPV-transfected cells. This increase in intracellular calcium concentration was dependent on the presence of extracellular calcium ion and was inhibited by treatment with gadolinium ion, a stretch-activated calcium channel blocker. Thus, these results indicate that ascidian egg HrTRPV is an osmotically sensitive TRP channel.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Expression and characterization of LTx2, a neurotoxin from Lasiodora sp. effecting on calcium channels

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels.     

Polymerase chain reaction cloning of L-type calcium channel sequences from the heart and the brain.

The sequences of the highly conserved S4 regions of voltage-sensitive ion channels were used to design oligonucleotide primers for the polymerase chain reaction. Specific fragments of the cDNA encoding L-type calcium channels from the heart, brain, and skeletal muscle were amplified and cloned. The nucleotide sequences of the cardiac and brain calcium channels obtained are identical over this region, and share 78% homology with the skeletal muscle calcium channel. Comparison of the predicted amino acid sequences of our clones with those of other calcium channels reveals unexpected patterns of conservation which suggest alternative exon use.     

Modified autonomic regulation in mice with a P/Q-type calcium channel mutation

Recent genetic analyses revealed an important association between P/Q-type channels and hereditary neurological disorders. The α1 subunit of P/Q-type channels is coded by a single CaV2.1 gene. Since calcium entry via neuronal calcium channels is essential for neurotransmission, P/Q-type channels may play an important role in cardiac autonomic neurotransmission. To elucidate the physiological importance of P/Q-type channels in autonomic nerve control, we used rolling Nagoya (tg^rol) mice, which have a mutation in the CaV2.1 gene and decreased P/Q-type channel currents with reduced voltage sensitivity.The tg^rol mice demonstrated unmodified expression of other calcium channel subunits. Electrocardiogram and echocardiographic analyses revealed decreased heart rate. Furthermore, ω-agatoxin IVA, a P/Q-type channel inhibitor, decreased heart rate and ejection fraction only in wild-type mice, thus suggesting a significant involvement of P/Q-type channels in chronotropic regulation. Atrium contraction analyses revealed a minor but significant role for P/Q-type channels in sympathetic and parasympathetic nerve regulation.     

Biochemical properties and subcellular distribution of an N-type calcium channel alpha 1 subunit.

A site-directed anti-peptide antibody, CNB-1, that recognizes the alpha 1 subunit of rat brain class B calcium channels (rbB) immunoprecipitated 43% of the N-type calcium channels labeled by [125I]omega-conotoxin. CNB-1 recognized proteins of 240 and 210 kd, suggesting the presence of two size forms of this alpha 1 subunit. Calcium channels recognized by CNB-1 were localized predominantly in dendrites; both dendritic shafts and punctate synaptic structures upon the dendrites were labeled. The large terminals of the mossy fibers of the dentate gyrus granule neurons were heavily labeled, suggesting that the punctate labeling pattern represents calcium channels in nerve terminals. The pattern of immunostaining was cell specific. The cell bodies of some pyramidal cells in layers II, III, and V of the dorsal cortex, Purkinje cells, and scattered cell bodies elsewhere in the brain were also labeled at a low level. The results define complementary distributions of N- and L-type calcium channels in dendrites, nerve terminals, and cell bodies of most central neurons and support distinct functional roles in calcium-dependent electrical activity, intracellular calcium regulation, and neurotransmitter release for these two channel types.     

Can presynaptic depolarization release transmitter without calcium influx?

Recent experimental evidence suggesting that presynaptic depolarization can evoke transmitter release without calcium influx has been re-examined. The presynaptic terminal of the squid giant synapse can be depolarized by variable amounts while recording presynaptic calcium current under voltage clamp and postsynaptic responses. Small depolarizations open few calcium channels with large single channel currents. Large depolarizations approaching the calcium equilibrium potential open many channels with small single channel currents. When responses to small and large depolarizations eliciting similar total macroscopic calcium currents are compared, the large pulses evoke more transmitter release. This apparent voltage-dependence of transmitter release may be explained by the greater overlap of calcium concentration domains surrounding single open calcium channels when many closely apposed channels open at large depolarizations. This channel domain overlap leads to higher calcium concentrations at transmitter release sites and more release for large depolarizations than for small depolarizations which open few widely dispersed channels. At neuromuscular junctions, a subthreshold depolarizing pulse to motor nerve terminals may release over a thousand times as much transmitter if it follows a brief train of presynaptic action potentials than if it occurs in isolation. This huge synaptic facilitation has been taken as indicative of a direct effect of voltage which is manifest only when prior activity raises presynaptic resting calcium levels. This large facilitation is actually due to a post-tetanic supernormal excitability in motor nerve terminals, causing the previously subthreshold test pulse to become suprathreshold and elicit a presynaptic action potential. When motor nerve terminals are depolarized by two pulses, as the first pulse increases above a certain level it evokes more transmitter release but less facilitation of the response to the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-throughput screening for N-type calcium channel blockers using a scintillation proximity assay

N-type calcium channels located on presynaptic nerve terminals regulate neurotransmitter release, including that from the spinal terminations of primary afferent nociceptors. Accordingly, N-type calcium channel blockers may have clinical utility as analgesic drugs. A selective N-type calcium channel inhibitor, ziconotide (Prialt), is a neuroactive peptide recently marketed as a novel nonopioid treatment for severe chronic pain. To develop a small-molecule N-type calcium channel blocker, the authors developed a 96-well plate high-throughput screening scintillation proximity assay (SPA) for N-type calcium channel blockers using [125I]-labeled omega-conotoxin GVIA as a channel-specific ligand. Assay reagents were handled using Caliper's Allegro automation system, and bound ligands were detected using a PerkinElmer TopCount. Using this assay, more than 150,000 compounds were screened at 10 microM and approximately 340 compounds were identified as hits, exhibiting at least 40% inhibition of [125I]GVIA binding. This is the 1st demonstration of the use of [125I]-labeled peptides with SPA beads to provide a binding assay for the evaluation of ligand binding to calcium channels. This assay could be a useful tool for drug discovery.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Leptinotarsin-D, a neurotoxic protein, evokes neurotransmitter release from, and calcium flux into, isolated electric organ nerve terminals

Previous work has demonstrated that the neurotoxin leptinotarsin elicits release of neurotransmitter from mammalian nerve terminals, and it has been suggested that the toxin may act either as a direct agonist of voltage-sensitive calcium channels in these terminals (Crosland et al., 1984) or as a calcium ionophore (Madeddu et al., 1985a,b). Preliminary studies (Yeager et al., 1987) demonstrated that leptinotarsin also evokes transmitter release from isolated elasmobranch electric organ nerve terminals. We now report further investigations of the effects of leptinotarsin in this system. The action of the toxin is saturable, releasing about the same small fraction of total transmitter as that released by depolarization. An upper limit for the concentration for half maximal release is estimated to be 4 nM. Leptinotarsin-evoked transmitter release exhibits behavior very similar to depolarization-evoked release with respect to dependence on Ca2+, Ba2+, and Sr2+ and blockade by Co2+, Cd2+, and trifluoperazine. Leptinotarsin also promotes the uptake of calcium into synaptosomes to a degree similar to that caused by depolarization by K+. The binding of leptinotarsin to nerve terminals is probably Ca2+ dependent and receptor mediated. Taken together with the behavior of leptinotarsin-evoked release in other preparations, these results are consistent with the hypothesis that this toxin acts by opening a presynaptic calcium channel. However, the possibility that leptinotarsin is a calcium ionophore cannot be excluded.     

Isolation, Cloning, and Characterisation of a trp Homologue from Squid (Loligo forbesi) Photoreceptor Membranes

Abstract: The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92-kDa component isolated from a detergent-insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full-length cDNA, encoding the 92-kDa component, using both cDNA library screening and 5'-rapid amplification of cDNA ends (5'-RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl-terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl-terminal domain has been shown to be in the regulation of several ligand-gated channels. The carboxyl-terminal domain has been expressed and shown to interact with calmodulin in a calcium-dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.     

Regulation of N-type Voltage-Gated Calcium Channels and Presynaptic Function by Cyclin-Dependent Kinase 5

N-type voltage-gated calcium channels localize to?presynaptic nerve terminals and mediate key events?including synaptogenesis and neurotransmission.?While several kinases have been implicated in the modulation of calcium channels, their impact on presynaptic functions remains unclear. Here we report that the N-type calcium channel is a substrate for cyclin-dependent kinase 5 (Cdk5). The pore-forming α(1) subunit of the N-type calcium channel is phosphorylated in the C-terminal domain, and phosphorylation results in enhanced calcium influx due to increased channel open probability. Phosphorylation of the N-type calcium channel by Cdk5 facilitates neurotransmitter release and alters presynaptic plasticity by increasing the number of docked vesicles at the synaptic cleft. These effects are mediated by an altered interaction between N-type calcium channels and RIM1, which tethers presynaptic calcium channels to the active zone. Collectively, our results highlight a molecular mechanism by which N-type calcium channels are regulated by Cdk5 to affect presynaptic function.     

Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

     

Antigens Associated with N- and L-Type Calcium Channels in Lambert-Eaton Myasthenic Syndrome

Abstract: In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (¹²⁵I-ω conotoxin GVIA) and L-type ([³H]PN200-110) calcium channels. Sera with a high antibody titer (>3 n M ) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.     

Acute axonal degeneration in vivo is attenuated by inhibition of autophagy in a calcium-dependent manner

Axonal degeneration is a pathological hallmark of many traumatic and neurodegenerative neurological disorders. Although the underlying mechanisms remain largely unclear, increased autophagy and the influx of extracellular calcium have been implicated in the pathogenesis of axonal degeneration based on in vitro data. Using in vivo imaging of the rat optic nerve after crush lesion we could now show that both mechanisms are linked and play an important role in acute axonal degeneration in vivo. Our data suggest that crush lesion of the optic nerve induces a rapid calcium influx through calcium channels, which results in a secondary induction of autophagy that participates actively in axonal degradation. Therapeutic manipulation of both events could significantly alter the time course of acute axonal degeneration in vivo and may thus represent promising therapeutic targets for the future.