Gap junctional channels comprised of connexin (Cx) subunits, commonly couple adjacent cells in mammalian tissue. We have shown that intracellular pH (pHi) modulates ventricular junctional conductance and permeability 1. A rise or fall of pHi from its resting level can be inhibitory. This influence was confirmed by measuring the pHi-sensitivity Cx43 channel conductance/permeability (the dominant ventricular isoform), with the protein heterologously expressed in cultured cell-lines 2. We have now investigated the possible molecular mechanism of this dual control by pHi. HeLa or N2a cell-pairs were stably transfected with Cx43. The open/closed status of Cx43 channels was assessed by two methods. (i) Cell-to-cell electrical conductance (gj) measured with dual whole-cell patch-clamp; pHi was displaced globally by acetate or trimethylamine [TMA] superfusion. (ii) Junctional H+ ion permeability (PHapp); H+ ions were UV-uncaged (from nitrobenzaldehyde) in one cell while confocally imaging (SNARF1) a pHi-decrease in the conjoined cell; pHi was adjusted to various starting levels before H+-uncaging, by pre-pulsing cells with weak acid (80mM acetate) or base (30mM NH4Cl). For the full-length Cx43 protein, gj started to change within 0.5min of a pHi displacement, stabilising in 4-8min. Displacing pHi from 7.1 to 6.4 or 7.3 reduced gj by 49±4% (n=7) and 55±30% (n=5), respectively. Corresponding PHapp values decreased by 77±10% (n=7, at pHi 6.6) and 80±7% (n=6, at pHi 7.3). Channel modulation by pHi was independent of any Ca2+i change (100μM AM-loaded BAPTA, n=7-13), and completely reversible: both gj and PHapp returned to initial values (in 5-10min) when pHi was returned to control levels.Cx43m257, a tailless mutant of Cx43 truncated at residue 257, lacked pHi-sensitivity. PHapp changed by-3±22% (n=8) and -9±10% (n=5) upon pHi displacement from 6.90 to 6.5 and 7.3 respectively. The gj value also remained unaltered during 40mM TMA perfusion and washout (n=3). The C-terminal cytosolic tail of Cx43 is thus involved in both high and low pHi block of the channel.An individual cysteine mutation, Cys298Ala, in the cytosolic tail, selectively removed Cx43 alkali-block, while preserving acid-block. PHapp decreased by 77±12% at pHi 6.6 (n=10) but remained unaltered at pHi 7.35 (+4±26%, n=17). Reducing pHi (80mM acetate, 4min) decreased gj by 58±7%, whereas raising pHi (40mM TMA, 4min) had no effect (n=5). We conclude that high and low pHi gating of Cx43 channels requires intact cytoplasmic tail regions of the protein. Selective removal of alkali-block by a single Cys mutation suggests that the two gates can operate independently.