1. Cell pairs of the insect cell line Sf9 (Spodoptera frugiperda) were chosen to examine the electrical properties of gap junction channels. The dual voltage-clamp method was used to control the membrane potential of each cell (V(m,1) and V(m,2)) and hence the junctional voltage gradient (V1), and to measure intercellular current. 2. Studies with preformed pairs revealed that the gap junction conductance (g(j)) is controlled by a V(j)- and a V(m)-sensitive gate. At steady state, g(j) = f(V(j)) was bell shaped and symmetrical (Boltzmann: V(j,0) = -54 and 55 mV, the normalized minimum conductance at large V(j) values (g(j,min) = 0.24 and 0.23, z = 5.5 and 6.1 for negative and positive V(j), respectively) and g(j) = f(V(m)) was S shaped (V(m,0) = 13 mV, g(j,main) = 0, z = 1.5). 3. Single channels exhibited two conductances, a main state (γ(j,main)) of 224 pS and a residual state (γ(j,residual)) of 42 pS. 4. We conclude that gap junctions in Sf9 cells behave similarly to those in the insect cell line C6/36 (Aedes albopictus). 5. An induced cell pair approach was used to examine heterotypic gap junction channels between Sf9 cells and C3/36 cells. 6. Heterotypic channels showed a γ(j,main) of 303 pS and a γ(J,residual) of 45 and 64 pS, depending on whether the Sf9 cell or C6/36 cell was positive inside. 7. In heterotypic gap junctions, g(j) = f(V(j)) was bell shaped and asymmetrical (g(j) was more sensitive to V(j) when the C6/36 cell was positive inside) and g(j) = f(V(m)) was S shaped (V(m,0) = 2 mV, g(j,min) = 0, z = 2.9). 8. We conclude that heterotypic channels possess a V(j)- and V(m)-sensitive gating mechanism. V(j) gating involves two gates, one located in each hemi-channel. V(j) gates are operated independently and close when the cytoplasmic aspect is made positive. 9. A comparison of homo- and heterotypic channel data suggests that docking of hemi-channels may affect channel gating, but not channel conductance.
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