Excitation of Skinned Muscle Fibers by Imposed Ion Gradients IlL Distribution of Permeant Ions in Unstimulated and Stimulated Fibers

Ion gradients imposed across an internal membrane system stimulate skinned muscle fibers; to evaluate the sarcoplasmic reticulum (SR) as the primary target site, SR polarization under resting and stimulatory conditions was assessed from fiber uptake o f permeant probe ions. Solvent spaces were estimated from simultaneous [14C]urea (U) or [SH]deoxyglucose (DOG) uptake in segments o f fibers from bullfrog semitendinosus muscle, skinned by microdissection. The distribution spaces, i.e., virtual solvent volumes at bath concentrations (Vu and Vtnx;), o f these uncharged probes correlated well with the protein content o f the same segments, which validated the tracer methodology for volume normalization. The membrane-bounded volume fraction (Vm), derived from the difference between total solvent volume (V,) and the non-membrane-bounded solvent volume (V~), was sufficient to detect appreciable SR ion accumulation. The V= estimated from the difference between Vv and Vm~6 assayed simultaneously with 2 or 5 -6 rain exposures was 10 I 1%, which is consistent with the morphometric volume fraction (mostly SR) in frog fibers; however, the change in this difference after membrane permeabilization corresponded to V= only 5%, The change in permeant ion distribution space caused by member permeabilization was used to assess SR membrane polarization, assuming the free ions distribute across the intact membrane according to the Nernst ratio. Resting polarization (SR lumen positive) was assessed from [~4C]SCNor [~4C]propionatedistribution spaces in unstimulated fibers, expressed relative to Vr~6 (assayed simultaneously). The ratios for (a) [t4C]SCNspace (carrier 2 raM) and (b) [14C]propionatespace (carrier 120 mM) were not decreased by membrane permeabilization. This indicated that anion distribution was independent of membrane integrity and did not reflect an SR transmembrane potential, although a was more and b was less than 1. Polarization Address reprint requests to Dr. Elizabeth Stephenson, Department of Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103-2757. J. Gzn. PHYSXOL. O The Rockefeller University Press 9 0022-1295/89/01/0151/21 $2.00 Volume 93 January 1989 151-171 151 on Jne 3, 2017 D ow nladed fom Published January 1, 1989