Intrinsic H+ ion mobility in the rabbit ventricular myocyte

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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 KCl 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 H+i movement was associated with a longitudinal intracellular pH (pHi) gradient of up to 0.4 pH units. H+i movement could be modelled using the equations for diffusion, assuming an apparent diffusion coefficient for H+ ions (DHapp) 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 H+i mobility is consistent with an average diffusion coefficient for the intracellular mobile buffers (Dmob) of ≈9 × 10−7 cm2 s−1.