Biophysical properties of the skeletal muscle L-type calcium channel in health and disease

The purpose of this project was three-fold: (a) to use electrophysiological measurements to gain a more detailed understanding of how the mutations causing Hypokalemic Periodic Paralysis affect the function of the skeletal muscle L-type Ca2+ channel, as a prerequisite to determining the sequence of events leading from the Ca2+ channel mutations to fiber paralysis; (b) to characterize a recently developed method for expressing this channel, which has resisted expression in non-muscle cells, in Xenopus laevis oocytes; and (c) to explore the gating process of the normal channel in detail. To accomplish these goals, a number of electrophysiological methods were used to record signals from muscle cells and Xenopus oocytes expressing normal and mutant channels.; In Chapter 1, patch clamp measurements of L-type Ca2+ current were made in normal and HypoPP (R528H) human myotubes in order to test for mutation-associated changes in activation and inactivation kinetics.; In Chapter 2, two electrode voltage-clamp (TEVC) recordings were made in Xenopus laevis oocytes expressing the cloned skeletal muscle L-type Ca2+ channel in order to study the effects of all three of the mutations in parallel.; In Chapter 3, the cut-open oocyte voltage clamp method developed by Stefani and Bezanilla (1998) was employed to measure gating charge movement in normal skeletal muscle L-type Ca2+ channels, and the significance of a muscle-specific post-translational truncation of the pore-forming alpha 1S subunit was explored. Substantial charge movement was measured, suggesting that oocyte expression of the channel is more robust than has been previously believed. The ratio of ionic current to gating current was more than six-fold higher for the truncated version of alpha1S than for the full-length version, suggesting that the full-length version may serve as a specialized voltage-sensor in muscle.; In Chapter 4, the cut-open oocyte method was applied to record gating charge movement from L-type channels carrying the HypoPP mutations in order to define the effects of the mutations on the early, rapid steps in channel gating. R528H reduced the amplitude of gating currents without shifting their voltage dependence, while R1239H both reduced the amplitude and shifted the voltage dependence of gating currents. R1239G had no effect on gating currents, despite its prominent effect on ionic currents. Thus, each of the mutations causing HypoPP had a distinct pattern of effects on the membrane expression, rapid charge movement, and slow channel gating of the dihydropyridine receptor, suggesting that fiber paralysis in HypoPP may arise from a diversity of underlying physiological defects. (Abstract shortened by UMI.)...