Intrinsic mobility of H+ ions (DHapp) in guinea pig ventricular myocytes

This was estimated by diffusing HCl (pH 3.0 in isotonic KCl) into a myocyte through a patch pipette while confocally imaging pHi (AM-loaded SNARF). Cells were superfused with 20 mM HEPES-(am-loaded SNARF). Cells were superfused with 20 mM HEPES-buffered Tyrode, pH 7.4 at 37°C. The time-course of fall of pHi was averaged in two regions of interest (ROIs) positioned 10 um and 105 um downstream from the pipette. Inter-ROI time delay, estimated at a 20 nM threshold rise of [H+]I, was 28s (n=14), and was unaffected (n=3) by 30 uM Hoe 642, a potent Na-H exchange inhibitor. Acid movement was associated with a longitudinal pHi gradient of about 0.25 units. Separate measurements of SNARFi mobility (n=4) indicated that protonated fluorophore movement accounted for <0.2% of the longitudinal acid flux. Modelling the spatiotemporal behaviour of pHi using equations for two-dimensional H+ diffusion indicated a DHapp of about 1.0 x 10-6 cm2/s, 2-3 fold faster than apparent in rabbit ventricular myocytes (Vaughan-Jones et al. Biophys J [2000] 78, 223A) but still 100 fold slower than for H+ in unbaffered solution. We conclude that effective intracellular H+ mobility is slow, most likely limited by the mobility of high capacity intrinsic buffers.