The alpha 2/delta subunit of voltage sensitive Ca2+ channels is a single transmembrane extracellular protein which is involved in regulated secretion.

The membrane topology of alpha 2/delta subunit was investigated utilizing electrophysiological functional assay and specific anti-alpha 2 antibodies. (a) cRNA encoding a deleted alpha 2/delta subunit was coinjected with alpha 1C subunit of the L-type calcium channel into Xenopus oocytes. The truncated form, lacking the third putative TM domain (alpha 2/delta delta TMIII), failed to amplify the expressed inward currents, normally induced by alpha 1C coinjected with intact alpha 2/delta subunit. Western blot analysis of alpha 2/delta delta TMIII shows the appearance of a degraded alpha 2 protein and no expression of the full-size two-TM truncated-protein. The improper processing of alpha 2/delta delta TMIII suggests that the alpha 2/delta is a single TM domain protein and the TM region is positioned at the delta subunit. (b) External application of anti-alpha 2 antibodies, prepared for an epitope within the alternatively spliced and 'intracellular' region, inhibits depolarization induced secretion in PC12, further supporting an external location of the alpha 2 subunit and establishing delta subunit as the only membrane anchor for the extracellular alpha 2 subunit.

Antibodies from ALS patients inhibit dopamine release mediated by L-type calcium channels.

OBJECTIVE: To examine the presence of anti-L-type calcium channel antibodies in the serum of ALS patients. BACKGROUND: Autoimmunity has been hypothesized as one of the mechanisms underlying the pathogenesis of sporadic ALS. Previous studies reported that sera from patients with sporadic ALS contain antibodies against voltage-gated calcium channels (L-type and P-type), but others do not support these findings. METHODS: Regulated secretion of tritiated dopamine ([3H]DA) in PC12 cells is mediated exclusively by calcium entry through L-type calcium channels. To examine whether purified ALS immunoglobulin G (IgG) inhibits [3H]DA release by interfering with calcium entry through L-type calcium channels, evoked release in PC12 cells was determined in the presence of ALS IgG. This functional assay provides a sensitive way to examine L-type calcium channel interaction with IgG from ALS patients. RESULTS: A significant inhibition of depolarization-evoked [3H]DA release (32+/-4%) was observed by purified IgG from ALS patients compared with control subjects (11+/-2%; p < 0.01). Significant inhibition by IgG occurred in 79% (15/19) of the ALS patients compared with only 29% (5/17) in the control group (p < 0.01). The level of calcium channel inhibition by ALS IgG correlated positively with disease duration (r = 0.68; p < 0.01) and correlated negatively with age (r = -0.48; p < 0.05). CONCLUSIONS: These results confirm the presence of antibodies against the L-type calcium channel in the majority of sera from ALS patients, supporting their role in the pathogenesis of ALS.

Identification of the alternative spliced form of the alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 cells.

The alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 has been cloned and partially sequenced. The message observed in Northern blot analysis displays a 7.5 kb transcript, identical in size to mRNA of rabbit skeletal muscle and rat brain. The nucleotide sequence of the cloned alpha 2 subunit of the PC12 specific cDNA is > 99% identical to rat brain sequence and 85% to skeletal muscle. Reverse-transcriptase-polymerase chain reaction (RT-PCR) of the alternative splicing region identifies two deleted regions of 57 bp and 21 bp in PC12 expressed alpha 2/delta transcript. The alternative variant alpha 2e of alpha 2/delta subunit which is expressed in PC12 cells was previously identified in human embryonic kidney (HEK293) cells. RT-PCR analysis show two different sized alternative PCR fragments in rat lung and none in rat spleen, kidney and intestine. Antibodies prepared against a 19 amino acid peptide within the alternative spliced region effectively inhibits [3H]dopamine release in PC12 cells. This implies that the alternatively spliced region is positioned extracellularly and is involved in regulation of the L-type Ca2+ channel-mediated transmitter release.

Characterization of the deg-3/des-2 receptor: a nicotinic acetylcholine receptor that mutates to cause neuronal degeneration.

The nicotinic acetylcholine receptor family (nAChR) is a large family of acetylcholine-gated cation channels. Here we characterize the Caenorhabditis elegans DEG-3/DES-2 nAChR, a receptor identified due to its involvement in neuronal degeneration. Pharmacological analysis of a DEG-3/DES-2 receptor expressed in Xenopus oocytes shows that this receptor is preferentially activated by choline. This choline sensitivity of the DEG-3/DES-2 channel can explain its role in neuronal degeneration, as shown by the toxic effects of choline on oocytes expressing the mutant DEG-3/DES-2 channel. We also show that in C. elegans the DEG-3/DES-2 receptor is localized to nonsynaptic regions, including the sensory endings of chemosensory neurons. This localization is in agreement with a role for this receptor in chemosensation of choline, as inferred from a defect in chemotaxis for choline seen in deg-3 mutants. Thus, this work also provides evidence for the diversity of nonsynaptic activities associated with nAChRs.