Dual Voltage-clamp Method Research Articles

Effects of phorbol ester on gap junctions of neonatal rat heart cells.

Myocytes were isolated from the ventricles of neonatal rat hearts and cultured for 1-3 days. Newly formed cell pairs were used to examine the conductance of gap junctions, gj. Measurements were performed using a dual voltage-clamp method in conjunction with a whole-cell, tight-seal recording. Exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 100-160 nM) led to a decrease in gj. Single-channel events recorded immediately before complete uncoupling yielded a single-channel conductance, gamma j, of 40.5 pS, implying that TPA affects the channel kinetics rather than gamma j. TPA-induced uncoupling was observed at subphysiological levels of cytosolic Ca2+ (pipette solution = 18 nM), not at physiological levels (pipette solution = 170 nM). The effects of TPA could not be mimicked by 250 microM 1-oleoyl-2-acetyl-glycerol (OAG). Preincubation with TPA (up to 24 h) revealed no changes in gj attributable to down-regulation of protein kinase C, PKC. Pretreatment with PKC inhibitors, staurosporine or PKCI, prevented the TPA-dependent decrease in gj. TPA-dependent uncoupling was not impaired by 4-bromophenacyl bromide, an inhibitor of phospholipase A2, PLA2; conversely, an arachidonic acid-dependent decrease in gj was not prevented by PKCI. This suggests that gj regulation does not involve an interaction between PLA2 and PKC.

Temperature dependence of gap junction properties in neonatal rat heart cells

Cell pairs of neonatal rat hearts were used to study the influence of temperature on the electrical properties of gap junctions. A dual voltage-clamp method was adopted, which allowed the voltage gradient between the cells to be controlled and the intercellular current flow to be measured. Cell pairs with normal coupling revealed a positive correlation between the conductance of the junctional membranes, gj, and temperature. Cooling from 37 degrees C to 14 degrees C led to a steeper decrease in gj, cooling from 14 degrees C to -2 degrees C to a shallower decrease (37 degrees C: gj = 48.3 nS; 14 degrees C: gj = 21.4 nS; -2 degrees C: gj = 17.5 nS), corresponding to a temperature coefficient, Q10, of 1.43 and 1.14 respectively. The existence of two Q10 values implies that gj may be controlled by enzymatic reactions. When gj was low, i.e. below 5 nS (conditions: low temperature; treatment with 3 mM heptanol), it showed voltage-dependent gating. This property was not visible when gj was large, i.e. 20-70 nS (conditions: high temperature; normal saline), presumably because of series resistances (pipette resistance). Cell pairs with weak intrinsic coupling and normally coupled cell pairs treated with 3 mM heptanol revealed a positive correlation between the conductance of single gap-junction channels, gamma j, and temperature (37 degrees C: 75.6 pS; -2 degrees C: 19.6 pS), corresponding to a Q10 of 1.41.