Modification of gap junction conductance by divalent cations and protons in neonatal rat heart cells

Myocytes were isolated from neonatal rat hearts and grown in culture dishes. Pairs of cells were selected to study the effect of divalent cations and protons on the conductance of gap junctions, g1. The experimental approach involved the dual voltage-clamp method and cell dialysis via patch pipette, i.e. g1 was monitored while the cytosolic level of Ca2+, Mg2+, Sr2+, Ba2+ or H+ was modified in one of the cells. A dose-dependent decrease in g1 developed when pCa of the pipette solution was lowered (range: pCa=7.7–2.42, equivalent to a [Ca2+] of 20 nm-3.8 mm). The g1/pCa-relationship revealed a Hill coefficient n of 0.87 and a half-maximal concentration pKCa of 3.5. Pretreatment with 3 mm NiCl2 and 1 μm ryanodine to minimize the removal of cytosolic Ca2+ did not significantly affect the response to g1. Similarly, g1 was decreased in a dose-dependent fashion when pHi in the pipette solution was lowered (range: pH=7.2−5.0, corresponding to a [H+] of 63 nm-10 μm). The g1/pH-relationship yielded an n of 0.92 and a pKH of 5.85. Pretreatment with 1 mm amiloride to minimize the extrusion of protons enhanced the effects of pH on g1. Simultaneous alterations in pCa and pH demonstrated an additive type of action of Ca2+ and H+ on g1. This is consistent with the existence of two types of sensors which contribute separately to the functional state of g1. No significant decrease in g1 was detectable when the pipette solution contained Mg2+ or Ba2+ (up to 5 mm). Partial uncoupling was observed with pipette solution containing 5 mm Sr2+. We conclude that g1 of neonatal and adult cardiomyocytes exhibit different ionic sensitivities. This discrepancy may reflect differences in connexin expression and/or molecular intermediates involved in regulating g1.