Intrinsic H+ ion mobility in the rabbit ventricular myocyte

Abstract

The intrinsic mobility of intracellular H+ ions was investigated by confocally imaging the longitudinal movement of acid inside rabbit ventricular myocytes loaded with the acetoxymethyl ester (AM) form of carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1). Acid was diffused into one end of the cell through a patch pipette filled with an isotonic KC1 solution of pH 3.0. Intracellular H+ mobility was low, acid taking 20-30 s to move 40 μm down the cell. Inhibiting sarcolemmal Na+-H+ exchange with 1 mM amiloride had no effect on this time delay. Net Hi+ movement was associated with a longitudinal intracellular pH (pHi) gradient of up to 0.4 pH units. Hi+ movement could be modelled using the equations for diffusion, assuming an apparent diffusion coefficient for H+ ions (DappH) of 3.78 × 10-7 cm2 S-1, a value more than 300-fold lower than the H+ diffusion coefficient in a dilute, unbuffered solution. Measurement of the intracellular concentration of SNARF (∼ 400 μM) and its intracellular diffusion coefficient (0.9 × 10-7 cm2 S-1) indicated that the fluorophore itself exerted an insignificant effect (between 0.6 and 3.3%)on the longitudinal movement of H+ equivalents inside the cell. The longitudinal movement of intracellular H+ is discussed in terms of a diffusive shuttling of H+ equivalents on high capacity mobile buffers which comprise about half (∼ 11 mM) of the total intrinsic buffering capacity within the myocyte (the other half being fixed buffer sites on low mobility, intracellular proteins). Intrinsic Hi+ mobility is consistent with an average diffusion coefficient for the intracellular mobile buffers (Dmob) of -e1 9 × 10-7 cm2 S-1.