GAP JUNCTION GATING SENSITIVITY TO PHYSIOLOGICAL INTERNAL CALCIUM REGARDLESS OF PH IN NOVIKOFF HEPATOMA-CELLS

Gap junction conductance (G(j)) and channel gating sensitivity to voltage, Ca2+, H+, and heptanol were studied by double whole-cell clamp in Novikoff hepatoma cell pairs. Channel gating was observed at transjunctional voltages (V(j)) > +/-50 mV. The cells readily uncoupled with 1 mM 1-heptanol. With heptanol, single (gap junctional) channel events with unitary conductances (gamma(j)) of 46 and 97 pS were detected. Both Ca2+-loading (EGTA.Ca) and acidifying (100% CO2) solutions caused uncoupling. However, CO2 was effective when Ca2+i was buffered with EGTA (a H+-sensitive Ca-buffer) but not with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (a H+-insensitive Ca-buffer), suggesting a Ca2+-mediated H+ effect on gap junctions. This was tested by monitoring the G(j) decay at different pCa(i) values (9, 6.9, 6.3, 6, and 5.5; 1 mM BAPTA) and pH(i) values (7.2 or 6.1, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2-(N-morpholino)ethansulphonic acid, respectively). With pCa(i) greater-than-or-equal-to 6.9 (pH 7.2 or 6.1), G(j) decreased to 10-70% of initial values in approximately 40 min, following single exponential decays (tau = approximately 28 min). With pCa(i) 6-6.3 (pH 7.2 or 6.1), G(j) decreased to 10-25% of initial values in 15 min (tau = approximately 5 min); the Student t gave a P = 0.0178. With pCa 5.5 the cells uncoupled in less than 1 min (tau = approximately 20 s). Low pH(i) affected neither time course nor shape of G(j) decay at any pCa(i) tested. The data indicate that these gap junctions are sensitive to [Ca2+]i in the physiological range (less-than-or-equal-to 500 nM) and that low pH(i), without an increase in [Ca2+]i, neither decreases G(j) nor increases channel sensitivity to Ca2+.