Proton Fluxes Across the Tubular (T-) System Membrane of Rat Fast-Twitch Fibres

Abstract

Cytoplasmic pH has major effects on most cellular processes in skeletal muscle, including its ability to develop force. Protons are continuously extruded from the cytoplasm against their electrochemical gradient as shown by the considerably more alkaline pH in the resting muscle than the predicted pH value if protons were distributed passively. We aimed to determine the contribution of the t-system proton extrusion mechanisms to this gradient and the diffusive proton flux of the t-system. To do this we trapped 10 mM of the pH-sensitive dye HPTS inside the tubular (t-) system of mechanically skinned fibres from the rat extensor digitorum longus muscles and continuously imaged dye fluorescence during changes in internal solution pH, [Na+], [K+] and [ATP] by confocal microscopy. Calibrations using monensin showed that in normally polarized fibres with 36 mM [Na+]cyto that pHt-sys was 7.50 ± 0.12 (n=3), 7.91 ± 0.20 (n=3) and 8.31 ± 0.29 (n=4) at pH-cyto of 6.8, 7.2 and 7.5, respectively. In the presence of 162 mM [Na+]cyto, with or without amiloride, the pHt-sys and [Na+]t-sys were similar to cytoplasmic values. The addition of 50 µM amiloride to normally polarized fibres with 36 mM [Na+]cyto increased pHt-sys further indicating that the Na+-H+ exchanger (NHE) was the major protein responsible for extruding protons from the cytoplasm. The pH difference across the t-system membrane at rest is reduced by NHE activity, which moves protons against the inward diffusive proton flux of the resting muscle fibre. We calculated the diffusive proton flux across the t-system to be 2.7 +/- 0.9 e-4 m/s (mean +/- SEM, n=3).