Prolonged depolarization promotes fast gating kinetics of L-type Ca2+ channels in mouse skeletal myotubes.
The effects of prolonged conditioning depolarizations on the activation kinetics of skeletal L-type calcium currents (L-currents) were characterized in mouse myotubes using the whole-cell patch clamp technique. The sum of two exponentials was required to adequately fit L-current activation and enabled determination of both the amplitudes (A(fast) and A(slow)) and time constants (tau(fast) and tau(slow)) of each component comprising the macroscopic current. Prepulses sufficient to activate (200 ms) or inactivate (10 s) L-channels did not alter tau(fast), tau(slow), or the fractional contribution of either the fast (A(fast)/(A(fast) + A(slow)) or slow (A(slow)/(A(fast) + A(slow))) amplitudes of subsequently activated L-currents. Prolonged depolarizations (60 s to +40 mV) resulted in the conversion of skeletal L-current to a fast gating mode following brief repriming intervals (3-10 s at -80 mV). Longer repriming intervals (30-60 s at -80 mV) restored L-channels to a predominantly slow gating mode. Accelerated L-currents originated from L-type calcium channels since they were completely blocked by a dihydropyridine antagonist (3 microM nifedipine) and exhibited a voltage dependence of activation similar to that observed in the absence of conditioning prepulses. The degree of L-current acceleration produced following prolonged depolarization was voltage dependent. For test potentials between +10 and +50 mV, the fractional contribution of Afast to the total current decreased exponentially with the test voltage (e-fold approximately 38 mV). Thus, L-current acceleration was most significant at more negative test potentials (e.g. +10 mV). Prolonged depolarization also accelerated L-currents recorded from myotubes derived from RyR1-knockout (dyspedic) mice. These results indicate that L-channel acceleration occurs even in the absence of RyR1, and is therefore likely to represent an intrinsic property of skeletal L-channels. The results describe a novel experimental protocol used to demonstrate that slowly activating mammalian skeletal muscle L-channels are capable of undergoing rapid, voltage-dependent transitions during channel activation. The transitions underlying rapid L-channel activation may reflect rapid transitions of the voltage sensor used to trigger the release of calcium from the sarcoplasmic reticulum during excitation-contraction coupling.
O'Connell
K M
KM
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Dirksen
R T
RT
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D046508:Culture Techniques; D004095:Dihydropyridines / Q000494:pharmacology; D004594:Electrophysiology; D015640:Ion Channel Gating; D007700:Kinetics; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D013997:Time Factors
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D019837:Ryanodine Receptor Calcium Release Channel; D009543:Nifedipine