Voltage-gated Ca2+ channels regulate the entry of Ca2+ ions into neurons and thereby control a variety of Ca2+-dependent processes such as neurotransmitter release, neurite outgrowth, excitability and gene expression. Electrophysiological experiments suggest that multiple types of Ca2+ channels (classified as T, N, L, and P) have evolved to perform specialized functions in different parts of the cell. The experiments outlined in this proposal are directed toward understanding the structural basis for this functional diversity. We have isolated overlapping cDNAs that code for the entire open reading frame of a novel Ca2+ channel (doe-4) from a library prepared from the electric lobe of the marine ray Discopyge ommata. Based on amino acid sequence analysis, doe-4 is approximately 40% homologous to skeletal and cardiac muscle L-type Ca2+ channels and approximately 75% homologous to a neuronal P-type channel. We have also cloned two other Ca2+ channels from the electric lobe, doe-2 and doe-3, that are nearly identical to the mammalian L-type and P-type channels, respectively. We suspect therefore that doe-4, the most abundant clone in the preparation, is an N-type Ca2+ channel. This is supported by our previous studies showing that Discopyge ommata electric organ is a rich source of binding sites for the N-type Ca2+ channel antagonist, (omegaCgTx. The overall goal of this project is to combine techniques in molecular biology and biochemistry to characterize the structural and functional properties of this important channel. We will use doe-4 cDNA as the starting material in a variety of experiments addressing structure-function questions. To characterize some of the structural properties of the channel, we propose to develop an array of anti-fusion protein antibodies directed against amino acid sequences specific to doe-4. Initial experiments will be aimed at determining the tissue distribution, subcellular localization, and subunit composition of the channel complex. We will also study the distribution properties of two alternatively spliced forms of the channel. The goal of these experiments is to gain a better understanding of the role that Ca2+ channels play in determining synaptic specialization. Moreover, we will study the functional properties of doe-4 expressed in a mammalian cell line. We will use antibodies as probes to determine the functional importance of various domains of the protein, and to study direct interactions with other subunits and regulatory proteins. We will also develop an assay for large scale screening of pharmacological compounds. An understanding of the molecular details of neuronal Ca2+ channel function will provide new insights into the complexities of neuronal excitability and neurotransmitter release, and may lead to the discovery of more effective therapy for treatment of a variety of neurotransmitter related disorders.
| Helton, Thomas D; Horne, William A (2002) Alternative splicing of the beta 4 subunit has alpha1 subunit subtype-specific effects on Ca2+ channel gating. J Neurosci 22:1573-82 |
