Membrane domain localization of ph-regulating transporters in frog
skeletal muscle membrane vesicles.
Putnam, Robert W., Phyllis B. Douglas, and N. A. Ritucci.
Department of Physiology and Biophysics, Wright State University,
School of Medicine, Dayton, Ohio 45435
APStracts 3:0159C, 1996.
The distribution of pH-regulating transporters in surface and
transverse tubular membrane (TTM) domains of frog skeletal muscle was
studied. BCECF-loaded giant sarcolemmal vesicles, containing surface
membrane, exhibited reversible Na+/H+ exchange. A microsomal vesicle
fraction was shown to be enriched in TTM based on high Na+/K+ ATPase
and Mg2+ ATPase activity, high ouabain and nitrendipine binding, and
low Ca2+ ATPase activity. TTM vesicles were well sealed and oriented
inside out. Vesicles were loaded with the pH-sensitive dye pyranine.
In response to an inwardly directed Na+ gradient, vesicles displayed
virtually no alkalinization unless monensin was present. No pH
response to an imposed Na+ gradient was seen regardless of the
direction of the pH gradient across the vesicles, after
phosphorylation of the vesicles with protein kinase C, or upon
exposure to GTP-_-S. In the presence of CO2, addition of Na+ or of
Cl- had no effect on vesicle pH. These data indicate that the
transverse tubular membrane lacks functional pH-regulating
transporters (Na+/H+ and (Na++HCO3-)/Cl- exchangers), suggesting that
pH-regulating transporters are localized only to the surface membrane
domain in frog muscle.
Received 8 August 1994; accepted in final form 26 April 1996.
APS Manuscript Number C478-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 28 May 96