Calcium ions enter cardiac cells through integral membrane proteins known as calcium channels, which behave as voltage-regulated gating devices. Within the cell, these ions modulate such essential events as contraction and automaticity. These studies propose to investigate the most basic elements of structure and function of the cardiac calcium channel. Two independent approaches will be used. 1). Isolation of the calcium channel protein. The first and most crucial step in this process is the identification of a ligand that is specific for the calcium channel protein. This may then be used as a tool in isolating the protein from the membrane for purification and peptide sequencing. Earlier studies from this laboratory have demonstrated the initial promise of atrotoxin, a component of venom from Crotalus atrox, as a specific ligand of the calcium channel protein. Studies proposed in this application will undertake identification of active subunits of atrotoxin and other snake venoms as a means for subsequent isolation and purification of the calcium channel protein. 2). Expression of the calcium channel protein in frog oocyte. Injection of messenger RNA from the nervous system of vertebrate organisms into the giant oocyte of Xenopus laevis, the South African clawed frog, induces the expression of functional ion channels in the oocyte membrane. This novel experimental preparation allows investigation of the physiology of normal channels and also those induced by mRNA in which site-specific mutation has occurred. Studies proposed herein will undertake the production of cardiac ion channels including the calcium channel in Xenopus oocyte. Identification of the responsible mRNA segment may allow selective expression of calcium channels, allowing a unique opportunity to study its physiology in the absence of other ion traffic. In future studies, we plan to employ genetic engineering determine the primary amino acid sequence of the channel protein once a selective mRNA is found. Determination of the calcium channel protein structure will allow better understanding of its function in health and disease.
| Moorman, J R; Zhou, Z; Kirsch, G E et al. (1987) Expression of single calcium channels in Xenopus oocytes after injection of mRNA from rat heart. Am J Physiol 253:H985-91 |
