Membrane domain localization of pH-regulating transporters in frog skeletal muscle membrane vesicles.
The distribution of pH-regulating transporters in surface and transverse (T) tubular membrane (TTM) domains of frog skeletal muscle was studied. 2',7'-Bis(carboxyethyl)-5(6)- carboxyfluorescein-loaded giant sarcolemmal vesicles, containing surface membrane, exhibited reversible Na+/H+ exchange. A microsomal vesicle fraction was shown to be enriched in TTM on the basis of high Na(+)-K(+)-ATPase and Mg(2+)-ATPase activity, high ouabain and nitrendipine binding, and low Ca(2+)-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 when exposed to guanosine 5'-O-(3-thiotriphosphate). In the presence of CO2, addition of Na+ or Cl- had no effect on vesicle pH. These data indicate that the TTM 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.
Department of Physiology and Biophysics, Wright State University, School of Medicine, Dayton, Ohio 45435, USA.
R W
RW
Putnam
P B
PB
Douglas
N A
NA
Ritucci
D000818:Animals; D001692:Biological Transport; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002474:Cell-Free System; D004077:Digoxin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis; D018482:Muscle, Skeletal / Q000502:physiology*; D009568:Nitrendipine / Q000494:pharmacology; D010042:Ouabain / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D011894:Rana pipiens; D012508:Sarcolemma / Q000502:physiology*; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D008024:Ligands; D009124:Muscle Proteins; D010042:Ouabain; D004077:Digoxin; D009568:Nitrendipine; D012964:Sodium; D011493:Protein Kinase C; D017301:Ca(2+) Mg(2+)-ATPase; D000254:Sodium-Potassium-Exchanging ATPase