CONDUCTANCES AND SELECTIVE PERMEABILITY OF CONNEXIN43 GAP JUNCTION CHANNELS EXAMINED IN NEONATAL RAT-HEART CELLS

Myocytes from neonatal rat hearts were used to assess the conductive p roperties of gap junction channels by means of the dual voltage-clamp method. The experiments were carried out on three types (groups) of pr eparations: (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 uncoupli ng agents such as SKF-525A (75 mu mol/L), heptanol (3 mmol/L), and ara chidonic acid (100 mu mol/L). In group 1, the first opening of a newly formed channel was slow (20 to 65 ms) and occurred 7 to 25 minutes af ter physical cell contact. The rate of channel insertion was 1.3 chann els/min. Associated with a junctional voltage gradient (V-j), the chan nels revealed multiple conductances, a main open state [gamma(j)(main state)], several substates [gamma(j)(substates)], and a residual state [gamma(j)(residual state)]. On rare occasions, the channels closed co m completely. The same phenomena were observed in groups 2 and 3. The existence of gamma(j)(residual state) provides an explanation for the incomplete inactivation of the junctional current (I-j) at large value s of V-j in cell pairs with many gap junction channels. The values of gamma(j)(main state) and gamma(j)(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 f or both conductances. The values of gamma(j) were dependent on the com position of the pipette solution. Solutions were as follows: (1) KCl s olution, gamma(j)(main state)=96 pS and gamma(j)(residual state)=23 pS ; (2) Cs+ aspartate(-) solution, gamma(j)(main state)=61 pS and gamma( j)(residual state)=12 pS; and (3) tetraethylammonium(+) aspartate(-) s olution, gamma(j)(main state)=19 pS and gamma(j)(residual state)=3 pS. The respective gamma(j)(main state)to-gamma(j)(residual state) ratios were 4.2, 5.1, and 6.3. This indicates that the residual state restri cts ion permeation more efficiently than does the main state. Transiti ons of I-j between open states (main open state, substates, and residu al state) were fast (<2 ms), and transitions involving the closed stat e and an open state were slow (15 to 65 ms). This implies the existenc e of two gating mechanisms. The residual state may be regarded as the ground state of electrical gating controlled by V-j; the closed state, as the ground state of chemical gating.