Skeletal muscle fibers from a patient with Schwartz-Jampel syndrome were studied in vitro. The fibers had normal resting membrane potentials, but their resting [Ca2+]i was elevated. The resting potentials were unstable and spontaneous depolarizations caused twitching in all fibers. Stimulated contractions were characterized by markedly slowed relaxation which was due to electrical after-activity. Neither curare (0.7 microM), tocainide (50 microM), nor phenytoin (80 microM) had an effect on the myotonic activity. In contrast, procainamide (200 microM) suppressed the hyperexcitability without affecting the twitch amplitude. The steady-state current-voltage relation was normal in 5 fibers, but altered in 3 others. These latter fibers had an increased specific membrane resistance owing to a decreased Cl- conductance. The Na+ channels were investigated in the cell-attached patch clamp mode. In all patches on either type of fiber, depolarizing pulses elicited delayed, synchronized openings of Na+ channels. These abnormal openings occurred even after the surface membrane repolarized. We hypothesize that these altered membrane conductances are responsible for the hyperexcitability and the associated slowed relaxation.
Ca(2+)-activated K+ channels of a large conductance (BKCa) in human skeletal muscle were studied by patch clamping membrane blebs and by using the three microelectrode voltage-clamp recording technique on resealed fibre segments. Single-channel recordings in bleb-attached and inside-out modes revealed BKCa conductances of 230 pS for symmetrical and 130 pS for physiological K+ distributions. Open probability increased with membrane depolarization and increasing internal [Ca2+]. The Hill coefficient was 2.0, indicating that at least two Ca2+ ions are required for full activation. Kinetic analysis revealed at least two open and three closed states. An additional long-lived inactivated state, lasting about 0.5-20 s, was observed following large depolarizations, when extracellular K+ was lowered to physiological values. BKCa were blocked by three means: (1) externally by tetraethylammonium which reduced single-channel amplitude (IC50 approx. 0.3 mM); (2) internally by polymyxin B which decreased the open probability (IC50 approx. 5 micrograms/ml); and (3) externally by charybdotoxin which caused long-lasting periods of inactivation (IC50 < 10 nM). Measurements on resealed fibre segments at physiological [K+] were in accordance with the single-channel data: only when intracellular [Ca2+] was elevated did charybdotoxin (50 nM) reduce the macroscopic membrane K+ conductance with depolarizing voltage steps.
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