Feliksas F. Bukauskas

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 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 micromol/L), heptanol (3 mmol/L), and arachidonic acid (100 micromol/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 [gamma(j)(main state)], several substates [gamma(j)(substates)], and a residual state [gamma(j)(residual state)]. On rare occasions, the channels closed 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 (Ij) at large values of Vj 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 for both conductances. The values of gamma(j) were dependent on the composition of the pipette solution. Solutions were as follows: (1) KCl solution, 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 solution, 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 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 (<2 ms), and transitions involving the closed state and an open state were slow (15 to 65 ms). This implies the existence of two gating mechanisms. The residual state may be regarded as the ground state of electrical gating controlled by Vj; the closed state, as the ground state of chemical gating.

Heterotypic gap junction channels (connexin26 – connexin32) violate the paradigm of unitary conductance

Human HeLa cells transfected with mouse DNA coding for connexin26 (Cx26) or connexin32 (Cx32) were used to examine the properties of heterotypic Cx26 – Cx32 gap junction channels. Intercellular current flow was examined in induced cell pairs by means of the dual voltage-clamp method. We found that Cx26 – Cx32 channels exhibit voltage-dependent conductances, γj: γj(main state) increases with increasing positivity at the cytoplasmic aspect of the Cx26 connexon and decreases with increasing negativity (slope: 32 pS/100 mV; γj γj(main state) reaches 48 pS as V j approaches 0 mV); γj(residual state) with a similar Vj-dependence is present when the cytoplasmic end of Cx26 connexon is positive, but absent when it is negative. The single channel data provide an explanation for the asymmetric relationships between the gap junction conductance, gj, and Vj. The results are consistent with the notion that docking of two connexons co-determines the biophysical properties of a gap junction channel.

Voltage-dependent gating of single gap junction channels in an insect cell line

De novo formation of cell pairs was used to examine the gating properties of single gap junction channels. Two separate cells of an insect cell line (clone C6/36, derived from the mosquito Aedes albopictus) were pushed against each other to provoke formation of gap junction channels. A dual voltage-clamp method was used to control the voltage gradient between the cells (Vj) and measure the intercellular current (Ij). The first sign of channel activity was apparent 4.7 min after cell contact. Steady-state coupling reached after 30 min revealed a conductance of 8.7 nS. Channel formation involved no leak between the intra- and extracellular space. The first opening of a newly formed channel was slow (25-28 ms). Each preparation passed through a phase with only one operational gap junction channel. This period was exploited to examine the single channel properties. We found that single channels exhibit several conductance states with different conductances gamma j; a fully open state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j(residual)) and a closed state (gamma j(closed)). The gamma j(main state) was 375 pS, and gamma j(residual) ranged from 30 to 90 pS. The transitions between adjacent substates were 1/7-1/4 of gamma j(main state). Vj had no effect on gamma j(main state), but slightly affected gamma j (residual). The lj transitions involving gamma j(closed) were slow (15-60 ms), whereas those not involving gamma j(closed) were fast (< 2 ms). An increase in Vj led to a decrease in open channel probability. Depolarization of the membrane potential (Vm) increased the incidence of slow transitions leading to gamma j(closed). We conclude that insect gap junctions possess two gates, a fast gate controlled by Vj and giving rise to gamma j(substates) and gamma j(residual), and a slow gate sensitive to Vm and able to close the channel completely.

Electrical coupling between cells of the insect Aedes albopictus.

1. Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used to study the electrical properties of intercellular junctions. A double voltage‐clamp approach was adopted to control the voltage gradient between the cells and measure the intracellular current flow. 2. Determinations of junctional conductance (gj) revealed two types of intercellular contacts, gap junctions and cytoplasmic bridges. Identification occurred by means of functional criteria, i.e. the dependency of gj on (i) junctional membrane potential, (ii) non‐junctional membrane potential, and (iii) heptanol. 3. In cell pairs with putative gap junctions, gj was dependent on the junctional membrane potential (Vj). When determined at the beginning of voltage pulses, gj was insensitive to Vj; when determined at the end of 15 s pulses, it depended on Vj in a bell‐shaped manner (70% decrease for a change in Vj of +/‐ 75 mV). 4. These cell pairs also showed a dependency of gj on the non‐junctional membrane potential (Vm). When determined immediately after changing the non‐junctional membrane potential in both cells, gj was not affected by Vm; when determined 30 s later, gj was modified by Vm in a S‐shaped fashion (100% decrease when Vm was depolarized to +50 mV). 5. Exposure to 3 mM‐heptanol gave rise to complete and reversible block of gj in cell pairs with putative gap junctions. 6. Cell pairs susceptible to uncoupling by heptanol revealed junctional currents indicative of the operation of gap junction channels. The single‐channel conductance, determined at a Vm of ‐50 to ‐70 mV, was 133 pS. 7. In the case of putative cytoplasmic bridges, gj was insensitive to the junctional and non‐junctional membrane potential. In addition, it was not affected by 3 mM‐heptanol. 8. While most cell pairs showed functional properties characteristic of gap junctions or cytoplasmic bridges, few cell pairs exhibited junctional currents compatible with the co‐existence of both junctional structures.

Multiple conductance states of newly formed single gap junction channels between insect cells

Two cells of an insect cell line (Aedes albopictus, clone C6/36) were pushed together to form a cell pair while the intercellular current flow was monitored. This approach enabled us to study the formation of gap junction channels and explore their electrical properties. We found that the single channels exhibit multiple conductance states. The conductance of a fully open channel was 365 pS; the subconductance steps were 1/7 to 1/5 of the maximal conductance. The voltage gradient across the junction did not influence the conductance of fully open channels, but affected the dwell time at particular conductance states. The latter provides an explanation for the voltage-dependent conductance of gap junction membranes seen in these cells. The very first channel opening always was slow (15–50 ms), suggesting the involvement of a mechanism different from conventional channel gating.

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° C to 14° C led to a steeper decrease in gj, cooling from 14° C to -2° C to a shallower decrease (37° C: gj=48.3 nS; 14° C: gj=21.4 nS;-2°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, γj, and temperature (37° C: 75.6 pS; -2°C: 19.6 pS), corresponding to a Q10 of 1.41.

Gap junction channels of insects exhibit a residual conductance

Formation of gap junction coupled cell pairs was used to assess the basic properties of single gap junction channels. For this purpose, two single cells (clone C6/36, derived from larvae of an insect, Aedes albopictus) were manoeuvred against each other to provoke gap junction channel insertion. Intercellular current flow was measured with a dual voltage-clamp method. Utilizing this approach, we were able to demonstrate that gap junction channels, after formation, do not close completely upon application of a transjunctional voltage gradient, Vj. Instead, they exhibit a residual conductance, γj(residual). On average, γj(residual) was 64±4 pS (n=40). This corresponds to about 1/6 of the conductance of a fully open channel. The existence of γj(residual) explains the observation that the conductance of the entire gap junction, gj, decreases only partially at large Vj.

Biophysical properties of heterotypic gap junctions newly formed between two types of insect cells.

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 (Vm,1 and Vm,2) and hence the junctional voltage gradient (Vj), and to measure intercellular current. 2. Studies with preformed pairs revealed that the gap junction conductance (gj) is controlled by a Vj‐ and a Vm‐sensitive gate. At steady state, gj = f(Vj) was bell shaped and symmetrical (Boltzmann: Vj.0 = ‐54 and 55 mV, the normalized minimum conductance at large Vj values (gj,min) = 0.24 and 0.23, z = 5.5 and 6.1 for negative and positive Vj, respectively) and gj = f(Vm) was S shaped (Vm.0 = 13 mV, gj,min = 0, z = 1.5). 3. Single channels exhibited two conductances, a main state (gamma j,main) of 224 pS and a residual state (gamma 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 gamma j,main of 303 pS and a gamma 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, gj = f(Vj) was bell shaped and asymmetrical (gj was more sensitive to Vj when the C6/36 cell was positive inside) and gj = f(Vm) was S shaped (Vm,0 = 2 mV, gj,min = 0, z = 2.9). 8. We conclude that heterotypic channels possess a Vj‐ and Vm‐sensitive gating mechanism. Vj gating involves two gates, one located in each hemi‐channel. Vj 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.