The biochemical and biophysical properties of voltage-dependent calcium channels in the membranes of excitable cells will be investigated. The overall goal is to determine the extent of functional homology of calcium channels in different tissues and to evaluate their importance in regulating physiological responses. It is proposed to prepare and characterize membrane vesicles enriched in calcium channel proteins from skeletal muscle, smooth muscle and brain and to investigate the mechanisms by which channel activity is regulated by membrane potential and drug binding. The role of calcium channels in neurotransmission will be studied by measuring the release of neurotransmitters from brain synaptosome preparations in response to voltage-dependent calcium uptake. Binding proteins for channel blocking drugs such as the 1,4- dihydropyridines have been identified in brain, cardiac, smooth and skeletal muscle. These binding proteins are likely to represent calcium channels but, for reasons that are not yet understood, the drugs do not appear to have significant effects on calcium channel function in skeletal muscle and brain. In order to investigate the molecular basis for these observations, parallel measurements will be made of the ligand binding and ion transporting properties of voltage-dependent calcium channels in membrane vesicles from rabbit skeletal muscle, rabbit ileal longitudinal smooth muscle and rat brain. Rapid kinetic techniques will be used to monitor calcium ion fluxes on timescales of physiological relevance. Ligand binding to membrane preparations will be measured under both equilibrium and pre-equilibrium conditions in order to identify transient conformational states of the channel protein that may be important in its function. The dihydropyridine receptor from skeletal muscle will also be purified and its functional integrity assessed after reconstitution into phospholipid vesicles. These measurements will allow a rigorous evaluation of the mechanisms involved in the regulation of calcium channel activity. In addition they will provide specific molecular information on the basis for the discrepancies that are frequently observed between the concentrations of ligands that influence calcium channel function and the much lower concentrations that are required to saturate the high-affinity binding sites measured in vitro.
| Oz, A M; Frank, G B; Dunn, S M (1993) Voltage-dependent calcium fluxes in skeletal muscle transverse tubule membranes in the range of late afterpotentials. Can J Physiol Pharmacol 71:518-21 |
| Dunn, S M; Bladen, C (1992) Low-affinity binding sites for 1,4-dihydropyridines in skeletal muscle transverse tubule membranes revealed by changes in the fluorescence of felodipine. Biochemistry 31:4039-45 |
| Dunn, S M; Bladen, C (1991) Kinetics of binding of dihydropyridine calcium channel ligands to skeletal muscle membranes: evidence for low-affinity sites and for the involvement of G proteins. Biochemistry 30:5716-21 |
