The broad, long-term objectives of the project is to understand the molecular interactions between presynaptic voltage-gated calcium (Ca2+) channels and modular adaptor proteins. Opening of voltage-gated N-type and P/Q-type Ca2+ channels results in Ca2+ influx into presynaptic terminal, synaptic vesicle fusion and secretion of a neurotransmitter. Preliminary biochemical experiments show that carboxy-terminal regions of N-type and P/Q-type Ca2+ channels specifically associate with mint1 and CASK proteins. Mint1 and CASK are modular adaptor proteins which participate in formation of recently identified presynaptic tripartite protein complex. The sites responsible for interaction of Q-type (a1A) Ca2+ channels with mint1 and CASK are localized in the immediate proximity to the position of polyglutamine repeat expansion in a1A sequence, which has been linked to autosomal dominant cerebellar ataxia (SCA6) disorder in humans. The uncovered ineractions between voltage-gated Ca2+ channels and adaptor proteins may potentially be important for functional regulation of these channels, for targeting of Ca2+ channels to axonal compartment and to the synapse and/or for proper localization of presynaptic Ca2+ channels relative to synaptic vesicle docking and fusion machinery. The proposal is focused on testing these ideas. During the grant support period, we intend: 1. To identify R-type (a1E) carboxy-terminal binding partners by yeast two-hybrid and biochemical methods. 2. To establish the role of N-type channel interactions with CASK is their modulation by G-proteins in biochemical experiments and in Xenopus oocytes functional expression system 3. To determine the functional significance of N-type channel interactions with Mint1 and CASK for synaptic transmission in superior cervical ganglion (SCG) neuronal cultures 4. To establish the significance of N-type channel interactions with Mint1 and CASK for channel targeting to axonal compartment and to synapse in CNS. GFP- and epitope-tagged Ca2+ channel constructs will be expressed in primary hippocampal neuronal cultures by adenovirus-mediated infection for imaging/localization experiments. The results of these experiments will provide essential new information required to understand function of Ca2+ channels in relation to synaptic transmission and may provide an insight into causes of SCA6 and other neurodegenerative diseases.
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