| Abstract No.: | C-B3056 |
| Country: | Canada |
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| Title: | EXPRESSION AND BIOPHYSICAL PROPERTIES OF NOVEL SPLICE VARIANTS OF RAT CAV2.1 |
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| Authors/Affiliations: | 2 Daoyi Wang*; 1 Jonathan Davies; 2 W.R.A. Kosala Rajapaksha; 1 Lina Chen; 1 Gerald Zamponi; 2 Thomas Fisher;
1 University of Calgary, AB, Canada; 2 University of Saskatchewan, Saskatoon, SK, Canada
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| Content: | Objectives: Voltage-gated Ca2+ channels mediate the Ca2+ influx that triggers exocytotic secretion. The synaptic protein interaction (synprint) site found in the intracellular II-III loop of CaV2.1 and CaV2.2 enables an association with synaptic proteins that may be crucial for neurotransmission and axonal targeting. Since II-III loop splice variants have been identified in CaV2.2, we characterized alternative splicing of the II-III loop of CaV2.1 and identified two splice variants, CaV2.1-Δ1 (which has a deletion of amino acids 754-948) and CaV2.1-Δ2 (which lacks amino acids 793-984). Our objectives are to determine whether these variants are expressed in neuroendocrine cells and to determine their electrophysiological properties in an exogenous expression system.
Materials and Methods: RT-PCR was performed on mRNA isolated from rat brain, magnocellular neurosecretory cells (MNCs), and PC12 cells. Antibodies directed against sequences in the CaV2.1 synprint site (which would recognize only the full length channel) and in the carboxyl terminus (which would also identify the deletion variants) were used to perform immunocytochemistry on cultured hippocampal neurons, MNCs, and PC12 cells. The biophysical properties of CaV2.1-Δ1 and CaV2.1-Δ2 were compared with wild-type CaV2.1 by co-expression of encoding plasmids with rat β1b, rat α2-δ1 and eGFP in tsA-201 cells, followed by whole cell patch clamp recordings.
Results: mRNA sequences coding for CaV2.1-Δ1 and CaV2.1-Δ2 were found in several brain tissues and in isolated MNCs and PC12 cells. Differences in immunolabeling of the two antibodies suggest that CaV2.1 II-III loop deletion variants are expressed in isolated MNCs and in cultured PC12 cells. Immunoreactivity for CaV2.1 splice variants was also found in the neuroendocrine terminals of the MNCs. The whole cell currents recorded in tsA-201 cells demonstrated that the current-voltage relationships for CaV2.1-Δ1 and CaV2.1-Δ2 were similar to those of wild-type CaV2.1, but that the peak current density was significantly smaller for the CaV2.1-Δ1 isoform. The steady state inactivation curve of the CaV2.1-Δ1 isoform was also shifted markedly toward more depolarized voltages.
Conclusions: Alternatively spliced variants of CaV2.1 that lack much of the synprint site are expressed in the somata of MNCs and PC12 cells and in the axon terminals of MNCs. This is consistent with the hypothesis that the interaction between synaptic proteins and CaV2.1 is important for synaptic transmission, but not for neuroendocrine secretion. The presence of a II-III loop splice variant in the MNC terminals suggests that axonal targeting in these cells does not require an interaction with synaptic proteins. Electrophysiological data indicate that one of these variants, CaV2.1- Δ1, has a marked depolarizing shift in its voltage dependence of inactivation, which may also reflect a functional difference in the channels mediating neuroendocrine secretion. Our data therefore have important implications for the role of the II-III loop in CaV2.1 function and targeting. |
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