The ability of vertebrate skeletal muscle to regulate intracellular pH (pHi) has received very little attention, despite the fact that changes in pHi can have a marked effect on muscle contraction and metabolism. Only in two muscles, the frog semitendinosus and the mouse soleus, have the membrane transport systems responsible for pH regulation been described. In the proposed research, three questions will be addressed with the aim of defining more fully the relationship between muscle cell function and pHi regulation. i) Are the pH-regulating systems in frog muscle (Na/H and (Na+HCO3)/C1 exchangers) localized to the surface membrane, the T tubular membrane or both? Localization of the transporters will be achieved by studying: pH recover (with glass electrodes) in glycerol-shocked fibers (whose T tubules vesiculate and are no longer in contact with the external medium); pH recovery in large vesicles made from surface membrane; Na uptake into small vesicles made from either surface or T tubular membrane; and acid extrusion into the T tubular lumen, using impermeable pH-sensitive fluorescent dyes. ii) Does acidification of unstirred layers, especially the T tubular lumen, limit the rate and extent of pHi recovery? The effect of unstirred layers will be assessed by measuring recovery in single vs. multicellular preparations and in fibers exposed to varying external buffer concentrations. The effect of acid extrusion on the pH in unstirred layers will be directly determined using pH-sensitive electrodes close to the fiber surface and using pH-sensitive fluorescent dyes in the T tubular lumen. iii) How does the ability to regulate pH differ in red vs. white muscle fibers? The buffering power, the rate and extent of pHi recovery, and the properties of the transport systems responsible for recovery will be compared in mouse soleus (red) and EDL (white) muscles. These studies will represent the first localization of a carrier system in muscle membrane, the first direct measurements of pH in the T tubular lumen and the first correlation of pH-regulating capabilities with cellular metabolic and contractile properties. Beyond the basic understanding of pH regulation gained in these studies, detailed knowledge of pH regulation may shed light on the etiology and/or symptoms of the muscular dystrophies and myotonias. One of the primary lesions of these diseases is an alteration of membrane properties which results in altered contractile and metabolic processes. The role that changes in pHi regulating ability may play in such pathology remains to be studied.