The Journal of General Physiology, Vol 93, 151-171, Copyright © 1989 by The Rockefeller University Press
Excitation of skinned muscle fibers by imposed ion gradients. III. Distribution of permeant ions in unstimulated and stimulated fibers
EW Stephenson
Department of Physiology, University of Medicine and Dentistry, New Jersey Medical School, Newark 07103.
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 of permeant probe ions. Solvent spaces were
estimated from simultaneous [14C]urea (U) or [3H]deoxyglucose (DOG) uptake
in segments of fibers from bullfrog semitendinosus muscle, skinned by
microdissection. The distribution spaces, i.e., virtual solvent volumes at
bath concentrations (Vu and VDOG), of these uncharged probes correlated
well with the protein content of 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
(Vs) and the non-membrane-bounded solvent volume (Vc), was sufficient to
detect appreciable SR ion accumulation. The Vm estimated from the
difference between VU and VDOG assayed simultaneously with 2 or 5-6 min
exposures was 10-11%, which is consistent with the morphometric volume
fraction (mostly SR) in frog fibers; however, the change in this difference
after membrane permeabilization corresponded to Vm 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 [14C]SCN- or
[14C]propionate- distribution spaces in unstimulated fibers, expressed
relative to VDOG (assayed simultaneously). The ratios for (a) [14C]SCN-
space (carrier 2 mM) and (b) [14C]propionate- space (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 under stimulatory conditions (lumen negative) was assessed
from 86Rb+ distribution, before and after an imposed ion gradient (choline
Cl replacement of K methanesulfonate (KMes) at constant [K+] [Cl-]) that
theoretically could generate a 48-fold transmembrane cation ratio; Ca
release was minimized by EGTA. The ratio of 86Rb+ space to VU, greater than
1 in KMes (120 mM K, the effective carrier), was higher in choline Cl (2.5
mM K) but not decreased by membrane permeabilization; this indicated that
86Rb+ distribution did not reflect an SR transmembrane potential. Similar
results in the presence of valinomycin ruled out the possibility of
inadequate 86Rb+ equilibration.(ABSTRACT TRUNCATED AT 400 WORDS)
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
