An improved vaseline gap voltage clamp for skeletal muscle fibers.

Abstract

A Vaseline gap potentiometric recording and voltage clamp method is developed for frog skeletal muscle fibers. The method is based on the Frankenhaeuser-Dodge voltage clamp for myelinated nerve with modifications to improve the frequency response, to compensate for external series resistance, and to compensate for the complex impedance of the current-passing pathway. Fragments of single muscle fibers are plucked from the semitendinosus muscle and mounted while depolarized by a solution like CsF. After Vaseline seals are formed between fluid pools, the fiber ends are cut once again, the central region is rinsed with Ringer solution, and the feedback amplifiers are turned on. Errors in the potential and current records are assessed by direct measurements with microelectrodes. The passive properties of the preparation are simulated by the disk equivalent circuit for the transverse tubular system and the derived parameters are similar to previous measurements with microelectrodes. Action potentials at 5.degree. C are long because of the absence of delayed rectification. Their shape is approximately simulated by solving the disk model with Na permeability in the surface and tubular membranes. Voltage clamp currents consist primarily of capacity currents and Na currents. The peak inward Na current density at 5.degree. C is 3.7 mA/cm2. At 5.degree. C the Na currents are smoothly graded with increasing depolarization and free of notches suggesting good control of the surface membrane. At higher temperatures a small, late extra inward current appears for small depolarizations that has the properties expected for excitation in the transverse tubular (T)-system. Comparison of recorded currents with simulations shows that while the T- system has regenerative Na currents, they are too small to make important errors in the total current recorded at the surface under voltage clamp at low temperature. The tubules are definitely not under voltage clamp control.