The goal of this project is to characterize, in detail and for the first time, the molecular properties of a presynaptic voltage- sensitive calcium ion channel protein. Neuronal calcium channels play an integral role in the processing and propagation of electrical signals within neurons and also initiate chemical synaptic transmission-perhaps the chief mechanism for signalling among neurons, sensory cells, and effector cells. changes in intracellular calcium concentration mediated by calcium channels are also likely to affect many other fundamental neuronal processes. These include ion channel permeability, cytoskeletal function, energy metabolism, catabolism, proliferation, and gene expression. Furthermore, calcium channels appear to be regulated by hormones and transmitters and, thus, are a potential molecular substrate for some aspects of the neuronal plasticity underlying learning, memory, and behavioral change. Their central role in nervous system function makes calcium channels both a possible locus of molecular deficit for one or more neuropathologies and a potential target for pharmacological intervention in a variety of neuropathologies. Purification and elucidation of the biochemical properties of a presynaptic voltage-sensitive calcium channel will provide insight into all these issues. Purification of calcium channels from nerve terminals of electric ray electric organ will be accomplished with the aid of two neurotoxic peptides: omega conotoxin and leptinotarsin. These toxins appear to bind specifically to electric organ calcium channels and will be used as affinity ligands to for identifying and purifying these channels from this synaptically-rich tissue. Purified channels will be functionally reconstituted in lipid bilayers to confirm that the purified toxin-binding protein is the calcium channel, to initiate investigations of channel electrical properties, and to assess potential regulation by hormones and transmitters through phosphorylation. In the long-term, purification will permit analyses of channel structure through chemical and enzymatic modifications, antibody binding, and cloning and sequencing of the channel gene(s). Since conotoxin and leptinotarsin binding sties are potential sites for regulation of channel function by novel pharmaceuticals, longer-term goals also include probing these binding sties by genetic manipulation of the amino acid sequences of both channel and conotoxin in combination with structural studies of these altered molecules. As a derivative benefit, the production of antibodies and complementary DNA will provide valuable probes to study both the regulation of expression and the physiological roles of calcium channels during development, nerve regeneration, and pathogenesis.
| Ahmad, S N; Miljanich, G P (1988) The calcium channel antagonist, omega-conotoxin, and electric organ nerve terminals: binding and inhibition of transmitter release and calcium influx. Brain Res 453:247-56 |
| Miljanich, G P; Yeager, R E; Hsiao, T H (1988) Leptinotarsin-D, a neurotoxic protein, evokes neurotransmitter release from, and calcium flux into, isolated electric organ nerve terminals. J Neurobiol 19:373-86 |
| Yeager, R E; Yoshikami, D; Rivier, J et al. (1987) Transmitter release from presynaptic terminals of electric organ: inhibition by the calcium channel antagonist omega Conus toxin. J Neurosci 7:2390-6 |
