William A. Horne - US grants

Normal cardiac function depends on the precise control of myoplasmic calcium concentration. The voltage-regulated calcium channel is an important part of this control mechanism. Cardiac disease such as hypertrophic cardiomyopathy, which is characterized by excessive intracellular Ca++ accumulation, may reflect a primary disorder in either calcium channel function, intracellular processing mechanisms, or calcium channel synthesis or degradation rates. These abnormalities may be expressed as changes in the number and distribution of channels within the cell membrane, or as a specific channel malfunction. To test this hypothesis, the functional properties of calcium channels in both normal and cardiomyopathic cells will be compared. Synthesis and degradation rates of the calcium channel and some aspects of its intracellular processing will be examined in both normal and cardiomyopathic cardiac cells. Cells will also be exposed to drugs known to influence calcium channel function (Beta-adrenergic agonists, Ca++ channel blockers) and the effects of these drugs on channel synthesis and degradation rates, and the number and distribution of channels on the cell will be determined. All parameters will be measured as a function of time in culture. Normal and cardiomyopathic (Syrian hamster BIO 14.6) heart cells will be cultured for study. Calcium channels will be identified and quantitated using the radioligand, [3H]nitrendipine. The affinity and number of binding sites will be determined by a filtration binding assay. The distribution of calcium channels ([3H]nitrendipine binding sites) on the cell surface will be determined by light microscopic autoradiography using tritium sensitive film. The functional properties of the channels will be characterized by measurement of the rate of 45Ca++ flux into the cells during K+ induced depolarization and by single channel recording using the patch clamp technique. The rates of synthesis and degradation of calcium channels will be determined by the density shift method which was developed by Devreotes et al. for measurement of acetylcholine receptor turnover rates. The role of glycosylation in intracellular processing will be examined using tunicamycin, an inhibitor of to protein glycosylation. The effects of cardiovascular drugs and several other manipulations on the number and turnover of calcium channels will be studied in order to determine if up- or down-regulation occurs with these treatments.

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.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how voltage-gated Ca2+ channels sense and decode electrical and molecular signals that regulate neurotransmitter release. Ca2+ channels are large (about 370 kDa) heteromultimeric proteins composed of alpha1, alpha2delta, and beta subunits that work in concert to control the amount of Ca2+ that enters a neuron in response to a given stimulus. The subunits form the Ca2+ channel pore (4 large transmembrane homology domains) and are encoded by at least ten genes that, based on structural, electrophysiological, and pharmacological differences, can be divided into three major sub-groups, Cav1, Cav2 and Cav3. Cav1 and Cav2 are high-voltage activating channels, whereas Cav3 channels activate at more negative membrane potentials. Cav1 genes express channels with L-type (long lasting) electrophysiological characteristics, Cav2 genes express P/Q, N, and R-type (intermediate lasting) channels, and Cav3 genes express T-type (transient) channels. The alpha1 subunits serve as targets for several classes of therapeutic agents, including antiarrhythmics (diltiazem, L-type antagonist) and analgesics (ziconotide, N-type antagonist from a marine snail), and for a host of peptide spider toxins (e.g., Aga IVA, P-type antagonist). Cav2 genes, which will be studied in this proposal, are expressed principally at synapses.The intracellular beta subunits, encoded by 4 distinct genes, interact with the alpha1 subunit at specific binding sites on between-homology-domain linker sequences. The beta subunits modulate Ca2+ channel expression levels, as well as the voltage dependence and kinetics of Ca2+ channel activation and inactivation. Our preliminary studies show that alternative splicing of the N-terminus of the beta4 subunit has alpha1 subunit subtype-specific effects on Ca2+ channel gating. They also show that splicing affects channel pharmacology (altered sensitivity to omegaCgTx GVIA) and responsiveness of alpha1 subunits to repetitive stimuli. Thus, understanding the molecular details of the events brought about by beta4 alternative splicing is essential for the development of analgesic drugs, and for furthering our understanding of the role that voltage-gated Ca2+ channels play in synaptic plasticity. To this end, our most remarkable preliminary result, obtained by using simple methods in structural genomics, is the discovery that the beta4 subunit and the synaptic scaffolding (MAGUK) protein, PSD-95, have evolved from a common ancestor. The two proteins share very similar predicted secondary structure, and with the crystal structure of PSD-95 now available, a number of beta4 subunit tertiary structure predictions can now be made. The objectives of this application are to confirm, using advanced NMR techniques, our tertiary structure predictions and to determine whether the well-characterized inter- and intramolecular interactions of PSD-95 have been conserved in beta4 subunits. Our hypothesis is that the beta4 subunit acts as a multi-modular docking site for a myriad of proteins, including calmodulin, kinase anchoring proteins, and PDZ domains, and serves as a director, transmitting molecular signals from inside the cell to the gating machinery of alpha1 subunits

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Small-angle X-ray Scattering of a protein complex formed by VGCC b4c and chromo shadow domain of HP1 A short splicing form of voltage-gated chalcium channel (VGCC) beta 4 subunit interacts with a nuclear protein Heterochromotin Protein 1 (HP1) and regulates gene silencing. The complex formation between the truncated VGCC b4 and chromo shadow domain (CSD) of HP1 has been confirmed in vitro by pull down experiment, ITC and NMR spectroscopy in Dr. Bill Horne[unreadable][unreadable]"s group in Vet school. Dr. Horne (PI) and his postdoc Dr. Xingfu Xu want to determine the molecular envelope of this complex using Small-angle X-ray scattering and validate the structure model derived from NMR docking.