Conductances and Selective Permeability of Connexin43 Gap Junction Channels Examined in Neonatal Rat Heart Cells

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Abstract
Myocytes from neonatal rat hearts were used to assess the conductive properties of gap junction channels by means of the dual voltage-clamp method. The experiments were carried out on three types (groups) of preparations: (1) induced cell pairs, (2) preformed cell pairs with few gap junction channels (1 to 3 channels), and (3) preformed cell pairs with many channels (100 to 200 channels) after treatment with uncoupling agents such as SKF-525A (75 μmol/L), heptanol (3 mmol/L), and arachidonic acid (100 μmol/L). In group 1, the first opening of a newly formed channel was slow (20 to 65 ms) and occurred 7 to 25 minutes after physical cell contact. The rate of channel insertion was 1.3 channels/min. Associated with a junctional voltage gradient (Vj), the channels revealed multiple conductances, a main open state [γj(main state)], several substates [γj(substates)], and a residual state [γj(residual state)]. On rare occasions, the channels closed completely. The same phenomena were observed in groups 2 and 3. The existence of γj(residual state) provides an explanation for the incomplete inactivation of the junctional current (Ij) at large values of Vj in cell pairs with many gap junction channels. The values of γj(main state) and γj(residual state) gained from groups 1, 2, and 3 turned out to be comparable and hence were pooled. The fit of the data to a Gaussian distribution revealed a narrow single peak for both conductances. The values of γj were dependent on the composition of the pipette solution. Solutions were as follows: (1) KCl solution, γj(main state)=96 pS and γj(residual state)=23 pS; (2) Cs+ aspartate− solution, γj(main state)=61 pS and γj(residual state)=12 pS; and (3) tetraethylammonium+ aspartate− solution, γj(main state)=19 pS and γj(residual state)=3 pS. The respective γj(main state)-to-γj(residual state) ratios were 4.2, 5.1, and 6.3. This indicates that the residual state restricts ion permeation more efficiently than does the main state. Transitions of Ij between open states (main open state, substates, and residual state) were fast (j; the closed state, as the ground state of chemical gating.