We have used whole-cell patch clamp recordings and pharmacological blockers of Ca channels to compare the pharmacology of Ca channels that mediate synaptic transmission at the three types of synapses innervating Purkinje cells in rat cerebellar slices. Both parallel fiber and climbing fiber excitatory synapses were sensitive to the P-type Ca channel blocker, ω-AgaIVA and the P/Q/N-type channel blocker, ω-conotoxin MVIIC. Transmission at inhibitory interneuronal synapses was not suppressed by these toxins, or by the N-type (ω-conotoxins GVIA and MVIIA) or L-type (nimodipine) channel blockers. Inhibitory transmission could be inhibited by Ni2+ and amiloride, but only at concentrations (IC50∼300 μM) that affect other types of Ca channels. These results indicate that excitatory and inhibitory presynaptic terminals of the cerebellar cortex possess different types of voltage-gated Ca channels. The excitatory terminals contain P-type, Q-type and N-type Ca channels, with P-type channels playing the most prominent role. The inhibitory terminals possess quite different type(s) of Ca channel. The heterogeneous distribution of Ca channel types should impart unique properties to transmitter release from the excitatory and inhibitory terminals. © 1997 Elsevier Science Ltd.

Voltage-sensitive Ca2+ channels are essential to transmitter release at the chemical synapse. To demonstrate the localization of voltage-sensitive Ca2+ channels in relation to the site of transmitter release, mouse neuromuscular junctions were double-labelled with alpha-bungarotoxin and a novel voltage-sensitive Ca2+ channel probe, SNX-260, a synthetic analog of omega-conopeptide MVIIC. Similar to omega-conopeptide MVIIC, biotinylated SNX-260 blocked nerve-stimulated transmitter release at the mouse neuromuscular junction. Fluorescently-tagged biotinylated SNX-260 labelled the nerve terminal which appeared thinner than and was outlined by acetylcholine receptor clusters as seen in en face view. This SNX-260 labelling was inhibited by preincubation with unconjugated SNX-260. Side-views of the neuromuscular junction indicated that the SNX-260 labelling was on the synaptic side facing the acetylcholine receptor rather than on the nonsynaptic side of the nerve terminal. This presynaptic binding was confirmed by the absence of SNX-260 labelling in denervated muscles following a nerve cut or disjunction after collagenase treatment. Confocal microscopy revealed spots of SNX-260 labelling that may correlate with active zones. The SNX-260 labelling pattern was not affected by preincubation with unconjugated SNX-111 (omega-conopeptide MVIIA), an N-type voltage-sensitive Ca2+ channel blocker. These findings suggest that SNX-260 is a novel probe for localizing non-N type voltage-sensitive Ca2+ channels and that these voltage-sensitive Ca2+ channels are localized near the transmitter release sites at the mammalian motor nerve terminal membrane. The results are consistent with the suggestion that non-N, probably P/Q type voltage-sensitive Ca2+ channels mediate evoked transmitter release at the mammalian neuromuscular junction.

BACKGROUND:Voltage-gated calcium channels in small-cell lung carcinomas may initiate autoimmunity in the paraneoplastic neuromuscular disorder Lambert-Eaton syndrome. The calcium-channel subtype that is responsible is not known.METHODS:We compared the effects of antagonists of L-type, N-type, and P/Q-type neuronal calcium channels on the depolarization-dependent influx of calcium-45 in cultured carcinoma cells. Serum samples from patients with various disorders were tested for reactivity with P/Q-type channels solubilized from carcinoma and cerebellar membranes and N-type channels from cerebral cortex.RESULTS:P/Q-type calcium-channel antagonists were the most potent inhibitors of depolarization-induced 45Ca influx in cultured small-cell carcinoma cell lines. Anti-P/Q-type calcium-channel antibodies were found in serum from all 32 patients with Lambert-Eaton syndrome and a diagnosis of cancer and in 91 percent of the 33 patients with Lambert-Eaton syndrome without cancer. Anti-N-type calcium-channel antibodies were found in 49 percent of the 65 patients with the Lambert-Eaton Syndrome. Lower titers of anti-P/Q-type and anti-N-type calcium-channel antibodies were found in 54 percent of 70 patients with a paraneoplastic encephalomyeloneuropathic complication of lung, ovarian, or breast carcinoma, 24 percent of 90 patients with cancer but no evident neurologic complications, 23 percent of 78 patients with sporadic amyotrophic lateral sclerosis, and less than 3 percent of 69 patients with myasthenia gravis, epilepsy, or scleroderma.CONCLUSIONS:The high frequency of P/Q-type calcium-channel antibodies found in patients with Lambert-Eaton syndrome implies that antibodies of this specificity have a role in the presynaptic pathophysiology of this disorder.

High-threshold voltage-sensitive calcium channels of the N-type, L-type, and P-type have been distinguished in the mammalian CNS predominantly on the basis of their sensitivity to selective antagonists. Matching them with genes identified by molecular cloning is an ongoing undertaking. Whereas L-type channels are characterize by their sensitivity to dihydropyridines and P-type channels by sensitivity to the funnel-web spider toxin AgaIVA, the N-type channel has been shown to be recognized by the ω-conopeptides GVIA and MVIIA. Recently, two new members of the family of ω-conopeptides—MVIIC from the marine snail Conus magus and SVIB from Conus strains—have been described. Binding and electrophysiological data suggest that these two peptides, in addition to interacting with N-type calcium channels, interact with a widely distributed receptor in neuronal membranes that is distinct from N-type channels. In this report we demonstrate through biochemical and pharmacological differentiation at individual receptor pelypeptide resolution, by affinity cross-linking, SDS-PAGE, and autoradiography, that SNX-230 (synthetic MVIIC) binds with high affinity to a calcium channel α1 subunit distinct from the high-affinity α1 target of SNX-111 (synthetic MVIIA). SNX-183 (synthetic SVIB) interacts with both α1 subunits with lower affinity. Whereas the α1 subunit recognized with high affinity by MVIIA corresponds to the N-type channel, the other represents a novel calcium channel distinct from N-, L-, and perhaps P-type channels.

The U1 snRNP-specific 70K protein is one of the few snRNP proteins from higher eukaryotic cells that is phosphorylated in vivo (1,2). Immunoaffinity purified spliceosomal snRNPs (U1, U2, U5, and U4/U6) were tested for their ability to phosphorylate in vitro the U1-specific 70K protein. An snRNP-associated kinase activity which phosphorylates all U1-70K isoelectric variants was identified. Like its in vivo counterpart, this snRNP-associated enzyme phosphorylates solely serine residues of the 70K protein, preferentially utilizing ATP as a phosphodonor. Tryptic phosphopeptide analysis revealed an overlapping set of at least four radiolabeled peptides in the in vivo and in vitro phosphorylated protein, suggesting that the snRNP-associated serine kinase is responsible, at least in part, for the 70K protein phosphorylation observed in vivo. Chymotryptic digestion of in vitro, 32P-labeled 70K protein and in vitro phosphorylation studies with a synthetic peptide, indicated that the multiple 70K phosphorylation sites are limited to a highly charged, C-terminal domain of the protein. In vitro phosphorylation studies with the splicing factor ASF/SF2 and several deletion mutants demonstrated that, similar to the U1-70K protein, the snRNP-associated serine kinase phosphorylates the carboxy terminal RS-rich domain of ASF/SF2. A potential general role for this enzyme in the phosphorylation of splicing factors and its consequences for pre-mRNA splicing regulation are discussed.

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.

Protein-RNA interactions in small nuclear ribonucleoproteins (UsnRNPs) from HeLa cells were investigated by irradiation of purified nucleoplasmic snRNPs U1 to U6 with UV light at 254 nm. The cross-linked proteins were analyzed on one- and two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage was demonstrated by isolating protein-oligonucleotide complexes from snRNPs containing 32P-labelled snRNAs after exhaustive digestion with a mixture of RNases of different specificities. The primary target of the UV-light induced cross-linking reaction between protein and RNA was protein F. It was also found to be cross-linked to U1 snRNA in purified U1 snRNPs. Protein F is known to be one of the common snRNP proteins, which together with D, E and G protect a 15-25 nucleotide long stretch of snRNAs U1, U2, U4 and U5, the so-called domain A or Sm binding site against nuclease digestion (Liautard et al., 1982). It is therefore likely that the core-protein may bind directly and specifically to the common snRNA domain A, or else to a sub-region of this. The second protein which was demonstrated to be cross-linked to snRNA was the U1 specific protein 70K. Since it has been shown that binding of protein 70K to U1 RNP requires the presence of the 5' stem and loop of U1 RNA (Hamm et al., 1987) it is likely that the 70K protein directly interacts with a sub-region of the first stem loop structure.