H+ ions, common end-products of metabolism, are potent modulators of cardiac function. H+ ions permeate at high flux through ventricular gap-junctions, providing one means for spatially equilibrating pHi within the myocardium. Gap-junctional H+-permeability & electrical-conductance are pH-gated, being acutely reduced by intracellular acid or alkaline-overload[1], a feature that regulates spatial H+i dispersion. We have now investigated the pH-sensitivity of connexin-43 gap-junctional channels, the dominant ventricular Cx-channel, stably transfected (rCx43, from rat) into HeLa and N2A cell-pairs, under conditions where surface membrane Na+/H+ exchange is inhibited with 30µM cariporide. Data are mean±SEM. Raising pHi from 7.0 to ~7.5 in 180s (20mM trimethylamine superfusion, TMA; pHi confocally imaged with SNARF-fluorophore) reversibly reduced cell-to-cell conductance (Gj; dual-cell voltage-clamp, -10mV hyperpolarising-step applied sequentially to each cell) by 53±7% in N2A pairs (n=5). Reducing pHi to <6.7 in 180s (80mM acetate superfusion) again reduced Gj (by 42±3%, n=6). Wild type (WT) cell-pairs lacking Cx43 exhibited no significant cell-to-cell conductance. Single channel conductance measurements (open conductance in 2 mM Halothane), were 122±10 pS, n=250, similar to previous values at normal and low pHi[2-3], suggesting that Gj-changes were caused by alterations in channel-gating kinetics. A pH-dependent Gj decrease was absent in N2A cell-pairs transfected with functional Cx43m275 mutant channel (Cx protein lacking much of original C-terminus, n=3), suggesting that the Cx cytoplasmic tail is necessary for both acid and alkali-gating. In other experiments, photolytic H+-uncaging from intracellular nitrobenzaldehyde (1mM)[4], within one cell of an rCx43 HeLa pair, promoted a pHi-fall (confocally imaged) in both cells, indicating junctional H+ permeation (again absent in WT cells lacking Cx43). Cell-to-cell H+ flux was large (up to 7.5mM/min for a cell-to-cell H+ gradient of <80nM; n=15). In some experiments, resting pHi was pre-adjusted (with TMA or acetate) before H+-uncaging. Apparent junctional H+-permeability estimated from the initial time course of H+-permeation, was reduced (by 88±12% and 93±5% n=4 & 6), when pHi in the junctional region was either increased or decreased (to 7.38 and 6.86 pHi respectively). Cell-to-cell H+ permeation was blocked by β-glycerrhetinic acid (60µM, selective Cx channel inhibitor; n=3), and was doubled (n=15) when 5%CO2/22mM HCO3- (plus 200µM DIDS) instead of 20mM Hepes-buffered superfusates were used. We conclude that Cx43 channels display characteristics of both high and low pH block, as previously identified for the ventricular gap junction. They are thus likely candidates for mediating spatial cell-to-cell pHi control in the myocardium.