Coupling asymmetry of heterotypic connexin 45/ connexin 43-EGFP gap junctions: properties of fast and slow gating mechanisms

Abstract

Although fast and slow gating mechanisms have been described in gap junctions (GJs), their relative contributions to dependence on transjunctional voltage, V(j), is still unclear. We used cell lines expressing wild-type connexin 45 (Cx45) and connexin 43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gating in homo- and heterotypic GJs formed of these connexins. Macroscopically Cx45/Cx45 channels show high sensitivity to V(j). Cx45 channels demonstrate two types of gating: fast transitions between open and residual states and slow transitions between open and completely closed states. Single-channel conductance of the Cx45 channel is approximately 32 pS for the open state and approximately 4 pS for the residual state. Cx45/Cx43-EGFP heterotypic junctions exhibit very asymmetrical V(j) gating with the maximum junctional conductance shifted to V(j) positive on the Cx45 side. Conductance of single Cx45/Cx43-EGFP channels is approximately 55 pS for the open state and approximately 4 pS for the residual state, values consistent with the simple-series connection of Cx45 and Cx43-EGFP hemichannels. At V(j) = 0, the slow gate of many Cx45 hemichannels is closed in both homotypic Cx45/Cx45 and heterotypic Cx45/Cx43-EGFP junctions. Fast and slow V(j) gates of both Cx45 and Cx43 hemichannels close for relative negativity at their cytoplasmic end. Coupling mediated by Cx45/Cx43-EGFP junctions can exhibit asymmetry that can be strongly modulated by small changes in difference of holding potentials. Cx45/Cx43 junctions are likely to be found in brain and heart and may mediate rectifying electrical transmission or modulatable chemical communication.

Figure 1

Macroscopic measurement of V_j gating of homotypic Cx45 junctions. (A) V_j gating in response to a long V_j step of −100 mV applied to cell 1. Repeated (1 Hz) V_j ramps (from −18 to +18 mV in 0.8 s) were applied before, during, and after the V_j step to monitor g_j. The g_j time plot determined from I_j and V_j records shows that during recovery from the step, g_j transiently exceeded the initial value; the maximum was termed g_jover. (B) Pooled data of normalized steady state g_j (G_j) vs. V_j measured in 15 Cx45 cell pairs. G_j was measured at the end of V_j steps ≈30 s in duration. The solid line is a fit of all of the data points by a four-state contingent gating model containing one gate per hemichannel (see below) with sensitivity parameters A = 0.3 mV⁻¹ and V₀ = 8.9 mV for negative V_j and A = 0.17 mV⁻¹ and V₀ = 7.5 mV. (C) Pooled data of normalized g_jover vs. V_j.

Figure 2

V_j gating of single Cx45 channels. (A) A negative and then a positive step of 60 mV were applied. At the beginning of the negative step a single channel was open (solid line) that showed closure to a substate of ≈4 pS (dashed lines), as well as a burst of apparently partial closings that were not well resolved. The closures were fast compared with the sampling interval of 2 ms. (B) Successive ramps repeated at 1/s showed fast gating to the residual state (first two ramps, dashed lines) and slow gating to the fully closed state (second two ramps, arrows). The channel was typically open at the onset of the ramp, closed at short latency, and then reopened (solid lines) at V_j approaching near zero.

Figure 3

V_j dependence of Cx45/Cx43-EGFP heterotypic junctions. (A) Phase contrast (Left) and fluorescence (Right) images of a Cx45/Cx43-EGFP cell pair. The Cx43-EGFP cell is identified by its fluorescence. The arrow indicates a junctional plaque. (B) Changes in I_j in response to positive and negative 60-mV V_j steps (positive V_j is defined as relatively positive on the Cx43-EGFP side). I_j increased slowly from an initial level of ≈13 pA during the negative V_j step and decreased rapidly during the positive V_j step. g_j decreased by about 30% between the steps. (C) Normalized g_j–V relation for Cx45/Cx43-EGFP heterotypic junctions in 14 cell pairs. The thin black line is a fit of a 4-state contingent gating model with one gate in each hemichannel to all data points (open and center-dotted circles). The parameters are A= 0.18 mV⁻¹ and V₀ = 1 mV for the Cx45 hemichannel and A = 0.03 mV⁻¹ and V₀ = 26 mV for the Cx43-EGFP hemichannel. The thick gray line is a fit of the data points indicated by dotted circles with a four-state contingent gating model of the channel containing fast and slow gates only in the Cx45 hemichannel. The parameters are A = 0.27 mV⁻¹ and V₀ = 3 mV for the slow gate and A = 0.46 mV⁻¹ and V₀ = 10 mV for the fast gate. Normalized g_j decreased rapidly with voltage for relative positivity on the Cx43-EGFP side; the relatively high sensitivity indicates that this decrease is mediated by the Cx45 hemichannel and that its gating polarity is negative. An expanded view (Inset) demonstrates that the contingent model can explain the secondary increase in g_j at large V_j (see Discussion and Fig. 6).

Figure 4

Asymmetric V_j gating in heterotypic Cx43/Cx43-EGFP junctions at the single-channel level. (A) Example of opening of Cx45/Cx43-EGFP channels during a V_j step of −60 mV applied to the Cx43-EGFP cell. Opening and closing transitions were between open and closed states. During a subsequent V_j step of +60-mV channels closed completely at short latency. (B) During negative V_j steps, the single channel gated between the open state with a conductance of ≈55 pS and the fully closed state. During positive pulses, the channel gated to the residual state (first pulse) or to the closed state (second and third pulses). (C) Ramps at 1 Hz from +100 to −100 mV show closure from the open state (solid lines) to the residual or to the closed state. As V_j became less positive, the channel opened and remained open during the negative part of the ramp.

Figure 5

Asymmetry of electrical coupling mediated by heterotypic Cx45/Cx43-EGFP junctions. (A) Asymmetry of electrical coupling in a Cx45/Cx43-EGFP cell pair. The Cx43-EGFP cell (V₁) was voltage clamped to positive or negative 100 mV, 30-ms rectangular pulses at 3 Hz from a holding potential of 0 mV. The Cx45 cell (V₂) was current clamped, and the steady-state holding potential was varied from ≈5 mV (from 0 to 18 s) to about −8 mV (from 18 to 62 s). Coupling was much greater for the negative pulses, and coupling asymmetry increased by making the Cx45 side more negative. (B) Schematics of cell pair; pulses were applied to HelaCx43-EGFP cell. (C) Pooled data of the electrical coupling asymmetry coefficient, K_asym, vs. ΔV_jh measured in seven Cx45/Cx43-EGFP cell pairs. Filled and open circles correspond to the experiments in which the Cx43-EGFP or Cx45 cell was voltage clamped and pulsed, respectively. Solid line shows data fit to sigmoid function, K_asym = A/{1 + exp[b(ΔV_jh − ΔV_jho)]}, with parameters A = 0.9338, b = 0.24 mV⁻¹, ΔV_jho = −6.4 mV.

Figure 6

Summary of the fit to the experimental data obtained in heterotypic Cx45/Cx43-EGFP junctions by a four-state contingent gating model. (A) V_j sensitivity of open probability of fast (dashed line) and slow (solid line) gates that operate only in the Cx45 hemichannel. (B) At V_j = 0 ≈70% of Cx45 hemichannels are closed solely by the slow gate (G_j normalized to value at V_j = 0 is shown by the thick gray line and P_o of the slow gate is shown by the solid line) and that the fast gate operates only at V_j values > ≈30 mV. (C and D) Heterotypic channels with identical (C) or different (D) conductances of hemichannels containing one slow gate (arrows) per hemichannel. (E) G_j–V_j dependence calculated for a four-state contingent gating model in which the two hemichannels have V_j sensitivity like that of Cx45 and Cx43 but are identical in unitary conductance that is equal to 230 pS (dashed line, C) or that they differ in both V_j sensitivity and unitary conductance (64 and 230 pS for the Cx45 and Cx43 hemichannels, respectively; solid line, D).