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, g(j). The experimental approach involved the dual voltage-clamp method and cell dialysis via patch pipette, i.e. g(i) 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 g(j) 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) mM). The g(j)/pCa-relationship revealed a Hill coefficient n of 0.87 and a half-maximal concentration pK(Ca) of 3.5. Pretreatment with 3 mM NiCl2, and 1 mu M ryanodine to minimize the removal of cytosolic Ca2+ did not significantly affect the response to g(j). Similarly, g(j) was decreased in a dose-dependent fashion when pH(i) in the pipette solution was lowered (range: pH = 7.2-5.0, corresponding to a [H+] of 63 nM-10 mu M). The g(j)/pH-relationship yielded an n of 0.92 and a pK(H), of 5.85. Pretreatment with 1 mM amiloride to minimize the extrusion of protons enhanced the effects of pH on g(i). Simultaneous alterations in pCa and pH demonstrated an additive type of action of Ca2+ and H+ on g(j). This is consistent with the existence of two types of sensors which contribute separately to the functional state of g(j). No significant decrease in g(j) 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 g(j) of neonatal and adult cardiomyocytes exhibit different ionic sensitivities. This discrepancy may reflect differences in connexin expression and/or molecular intermediates involved in regulating g(j). (C) 1995 Academic Press Limited