Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea‐pig.

1. The dependence of gap junctional conductance on the intracellular concentrations of H+, Ca2+ and Mg2+ was studied in paired myocytes dissociated enzymatically from guinea‐pig ventricle. To apply an internal solution buffered to specific H+, Ca2+ or Mg2+ concentration directly to one aspect of the gap junction, the non‐junctional membrane of one of the pair was mechanically ruptured. The junctional conductance was measured by clamping the membrane potential of the other cell using a two‐pipette voltage‐clamp method. 2. The conductance of the non‐junctional membrane was kept low in comparison with that of the junctional membrane (less than 1/50) by replacing both external and internal K+ with Cs+. 3. The current‐voltage (I‐V) relation of the junctional conductance was linear over the potential range examined (from ‐100 to +100 mV). No voltage or time dependence was detected. 4. The conductance of the gap junction between the paired cells ranged from 90 to 3900 nS with a peak distribution at 1000 nS. 5. The effect of H+ was examined over the pH range 7.4‐5.4, while keeping the free‐Ca2+ concentration at zero, or pCa 6.3 or 7.0 using 2‐10 mM‐EGTA. The junctional conductance was almost constant from pH 7.4 to 6.5 and decreased in a dose‐dependent manner with further acidification. There was no difference in the pH‐conductance relationships at various Ca2+ concentrations. The Hill coefficient was approximately 2.4 and the half‐maximum concentration (pK'H) was 6.1. 6. The closing effect of Ca2+ on the gap junction channel was examined over the concentration range from pCa 7 to 5, while keeping the pH at 7.4, 7.0 or 6.5. At each pH, increasing Ca2+ decreased the junctional conductance with similar Hill coefficients of about 3.4. The pCa‐conductance relationship shifted toward a higher Ca2+ concentration range as the pH was lowered (pK'Ca = 6.6, 6.4 and 5.6, at pH 7.4, 7.0 and 6.5, respectively). 7. Increasing Mg2+ also caused a fall in the junctional conductance over the pMg range 3.0‐2.0 with a pK'Mg of 2.5 (3.2 mM), and a Hill coefficient of 3.0. 8. These results suggest that there are two respective binding sites for divalent cations and H+, and that the gap junctional conductance is regulated reversibly by the ligand‐receptor reactions. Comparing the threshold concentrations of Ca2+ and H+ for electrical uncoupling, it was concluded that Ca2+ plays a more important role in regulating the gap junctional conductance of cardiac cells under physiological conditions.