Gap junction gating sensitivity to physiological internal calcium regardless of pH in Novikoff hepatoma cells.
Gap junction conductance (Gj) 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 (Vj) > +/- 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 Ca(2+)-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 Ca(2+)-mediated H+ effect on gap junctions. This was tested by monitoring the Gj decay at different pCai values (9, 6.9, 6.3, 6, and 5.5; 1 mM BAPTA) and pHi values (7.2 or 6.1, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2-(N-morpholino)ethansulphonic acid, respectively). With pCai > or = 6.9 (pH 7.2 or 6.1), Gj decreased to 10-70% of initial values in approximately 40 min, following single exponential decays (tau = approximately 28 min). With pCai 6-6.3 (pH 7.2 or 6.1), Gj 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 pHi affected neither time course nor shape of Gj decay at any pCai tested. The data indicate that these gap junctions are sensitive to [Ca2+]i in the physiological range (< or = 500 nM) and that low pHi, without an increase in [Ca2+]i, neither decreases Gj nor increases channel sensitivity to Ca2+.
Lazrak
A
A
Department of Physiology, University of Rochester, New York 14642-8642.
Peracchia
C
C
D000438:Alcohols / Q000494:pharmacology; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives; D004553:Electric Conductivity; D017097:Electric Impedance; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D019850:Heptanol; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008564:Membrane Potentials; D051381:Rats; D012996:Solutions; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000438:Alcohols; D002021:Buffers; D012996:Solutions; D004533:Egtazic Acid; D019850:Heptanol; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium