Long splice variant N type calcium channels are clustered at presynaptic transmitter release sites without modular adaptor proteins.
The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.
Cellular and Molecular Biology Division, Toronto Western Research Institute, University Health Network, 399 Bathurst Street, MP14-320, Toronto, Ontario, Canada M5T 2S8.
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Stanley
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002642:Chick Embryo; D019204:GTP-Binding Proteins / Q000378:metabolism; D005726:Ganglia, Parasympathetic / Q000378:metabolism; D051528:Guanylate Kinases; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D013347:Subcellular Fractions; D013570:Synaptic Membranes / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D020864:Calcium Channels, N-Type; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D020033:Protein Isoforms; C107293:Rim protein, mammalian; C106868:CASK kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D051528:Guanylate Kinases; D019204:GTP-Binding Proteins