| Abstract No.: | B-B2045 |
| Country: | Canada |
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| Title: | THE ROLE OF BETA SUBUNITS IN THE MEMBRANE TRAFFICKING OF CAV2 CHANNELS |
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| Authors/Affiliations: | 1 Taylor Dawson*; 1 Phuc Pham; 1 Adriano Senatore; 1 Patrick McCamphill; 1 J. David Spafford;
1 University of Waterloo, ON, Canada
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| Content: | Introduction: In synapses, P/Q-type and N-type Cav2 channels are stabilized in a dense protein matrix, clustered in proximity with the presynaptic machinery, where they control neurotransmitter release. To date, there is no consensus on required constituents or a mechanism for the membrane targeting of Cav2 channels in developing neurons per se. Previous work suggests that accessory β subunits may have a critical role to play in the membrane targeting of Cav2 channels (Spafford et al. 2004 J. Biol. Chem. 279:41157-41167.). Snail Cav2 channels appear to co-localize with β subunits in synaptically-paired neurons, but that the sole β subunit of snail calcium channels (LCavβ) is not coupled to Cav2 channels in new outgrowth of neurons. This has lead to a unique proposition that LCavβ promotes the membrane localization of Cav2 channels in growth cones.
Objectives: The objective of our research is to explore the hypothesis that there are motifs in the invertebrate β subunit which direct the membrane localization of Cav2 channels.
Materials and Methods: Our laboratory has screened fresh cDNA and cDNA libraries for novel splice variants of LCavβ and begun to assess the functional consequence of these beta subunit isoforms on LCav2 channels by whole-cell patch clamp recording of transfected subunits in mammalian HEK293T cells. We also have created probes for tracking LCav2 channels in vitro and in vivo, such as: a) an LCav2 channel incorporated with a 3x hemagglutinin (HA) extracellular epitope to track the membrane trafficking of heterologously-expressed LCav2 channels; b) We have worked in collaboration to probe for native Cav2 channels in developing growth cone membranes with a specific, extracellular epitope antibody (Zhang et al. 2008, J. of Neuroscience, in press).
Results: Using traditional screening methods, we have identified novel isoforms of the LCavβ including LCavβA1 and LCavβA2 which serve as mutually-exclusive, alternatively-spliced isoforms that differs in the first 49 and 47 amino acids of their N-termini respectively. LCavβA1 and LCavβA2 each have unique 5’ untranslated regions extending the unique sequences of each isoform, respectively to 312 bp and 345 bp. We have found that these different DNA sequences provide unique hybridization probes for labelling of genomic Southern blots and in situ hybridization. When expressed in vitro, LCavβA2 but not LCavβA1 will promote the membrane expression of snail LCav2 and alter its biophysical properties. Our data suggest that the 47 amino acid N-terminus of LCavβA2 is a key requirement for the membrane targeting of LCav2 channels.
Conclusion: So far our preliminary work indicates that a specific isoform of the N-terminus of the β subunit contains an important factor in the membrane trafficking of Cav2 channels in vitro. Our next set of experiments will be to assess the functional consequence of this alternative-spliced β subunit isoform on the recruitment of Cav2 channels to the membranes of developing growth cones. This research is important in the wider goal to model significant factors that contribute to the complex assembly of synapses. |
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