Gap junction channel gating

Abstract

Over the last two decades, the view of gap junction (GJ) channel gating has changed from one with GJs having a single transjunctional voltage-sensitive (V(j)-sensitive) gating mechanism to one with each hemichannel of a formed GJ channel, as well as unapposed hemichannels, containing two, molecularly distinct gating mechanisms. These mechanisms are termed fast gating and slow or 'loop' gating. It appears that the fast gating mechanism is solely sensitive to V(j) and induces fast gating transitions between the open state and a particular substate, termed the residual conductance state. The slow gating mechanism is also sensitive to V(j), but there is evidence that this gate may mediate gating by transmembrane voltage (V(m)), intracellular Ca(2+) and pH, chemical uncouplers and GJ channel opening during de novo channel formation. A distinguishing feature of the slow gate is that the gating transitions appear to be slow, consisting of a series of transient substates en route to opening and closing. Published reports suggest that both sensorial and gating elements of the fast gating mechanism are formed by transmembrane and cytoplamic components of connexins among which the N terminus is most essential and which determines gating polarity. We propose that the gating element of the slow gating mechanism is located closer to the central region of the channel pore and serves as a 'common' gate linked to several sensing elements that are responsive to different factors and located in different regions of the channel.

Fig. 1

Schematic representation of a GJ channel with presumed isopotential lines when both cells are held at the V_j = 0 mV (A), and at V_j = 100 mV (B & C) but at different values of V_m in each cell. In (A) the channel lumen is isopotential with cytoplasms of both cells; V_m1 = V_m2 = − 50 mV. This condition establishes a strong electric field (E) or a high density of isopotential lines across the channel wall in its central region. No V_j is established and E = 0 along the channel pore. GJ channels that respond to this voltage profile are termed V_m-sensitive. In (B), V_m1 differs from V_m2 establishing a V_j and a constant E along the pore; V_m changes along the channel pore from − 100 to 0 mV. In (C), the same V_j and profile of E along the channel pore are established as in (B), but with different values of V_m1 ( − 50 mV) and V_m2 (50 mV). GJ channels that respond the same way to voltage profiles in (B) and (C) are termed V_j-sensitive but not V_m-sensitive.

Fig. 2

Voltage gating in HeLaCx43 and Novikoff cell pairs. (A) Pooled data of normalized g_j vs. V_j measured in Novikoff (open circles) and HeLaCx43 (filled circles) cell pairs. The solid line is a fit of all the points to a Boltzmann relation with the following parameters: V₀ = 51 mV, A = 0.08 mV^{− 1} and G_min = 0.2 for negative V_js and V₀ = 50 mV, A = 0.09 mV^{− 1} and G_min = 0.18 for positive V_js. Squares filled with lines of different orientations indicate areas in which V_j gating is mediated predominantly through the slow gate (rising lines), the fast gate (declining lines) and both gates (crossing lines). (B) Schematic drawings of g_j evaluated over time in response to V_j steps varying in amplitude from ~ 30 to 150 mV that exemplify our findings in cells expressing Cx43 (see Fig. 1 in Ref. [51]). When V_j steps are < 50 mV (see g_j and V_j traces in 0–60-s interval), g_j declines and recovers slowly. When V_js are in the range of ~ 50–100mV(see g_j and V_j traces in 50–150-s time interval), g_j declines fast reaching steady state in few seconds. Recovery of g_j is also fast. At V_j > 100 mV, the decline in g_j is very fast initially and continues to decline more slowly without reaching steady state in the time interval shown (see g_j and V_j traces in 150–220-s interval).

Fig. 3

g_j–V_j dependence of Cx43 at the single channel level. (A) V_j steps applied to individual cell pairs. (B–E) I_j responses to V_j steps of 37 mV (B), 69 mV (C), 75 mV (D) and 105 mV (E). With a V_j step of 37 mV (B), four channels open at the beginning of the step and two closing transitions occur between open and closed states of ~ 110 pS (arrows) during the duration of the step. Also evident are several brief transitions, ~ 85 pS in magnitude (asterisks) representing transitions to the residual conductance state (γ_res). An expanded time scale (inset; sampling interval 1 ms) shows that the ~ 110-pS transitions are slow, taking several milliseconds to fully close the channel. At V_j = 69 mV (C), I_j declines rapidly through stepwise transitions of 85 pS indicating that the decline in g_j is via gating to γ_res. One channel undergoes a full 110-pS closing transition (first arrow). Also evident is a small 30-pS slow transition ascribable to a full closure of a channel from γ_res (second arrow; also see inset, sampling interval, 5 ms). At V_j = 75 mV (D), all the channels rapidly close to the residual state with transitions of 85 pS. At V_j = 107 mV (E), I_j declines very rapidly to a level that corresponds to all channels residing in γ_res and is followed by a slow decline in I_j through stepwise 30-pS transitions corresponding to full channel closures from γ_res. The expanded time scale (inset; sampling interval, 2 ms) shows the 30-pS transitions to be slow, taking several milliseconds to complete. Adapted from Ref. [51].

Fig. 4

Frequency histograms of I_j transition time between open and residual states (A) and between open and closed states (B). Data were collected from fibroblasts and HeLa-43 cells at V_js ranging from 25 to 75 mV during transient uncoupling by bath application of CO₂. Solid lines are Gaussian curves that fit to the data with mean values of 11 ± 0.8 ms (n = 264) and 2.0 ± 0.2 ms (n = 105) for upper and bottom plots, respectively. Adapted from Ref. [50].

Fig. 5

Voltage gating of wild-type Cx43 differs from that of Cx43-EGFP. (A –B) Single GJ channel gating transitions at V_j = 50 mV for Cx43 (left) and at V_j = 80 mV for Cx43-EGFP (right) expressed in HeLa cells. For Cx43, gating transitions are between open (solid line) and residual (dashed line) conductance states; full closures (not shown) occurred but were infrequent. For Cx43-EGFP, gating transitions are only between open and fully closed states. (C) Asymmetric V_j gating in heterotypic Cx43/Cx43-EGFP junctions. Shown are currents recorded from a cell pair containing two functional channels in response to a ± 100-mV ramp and ± 80-mV V_j steps applied to the HeLaCx43-EGFP cell. For the − 80-mV V_j step, only gating transitions of ~ 110 pS between open and closed states are observed, whereas for the + 80-mV V_j step, gating transitions of ~ 85 pS occur between open and residual states (dashed lines). Adapted from Ref. [51].

Fig. 6

Cx43-EGFP channels are permeable to negatively (Alexa fluor^{(1 − )}, Lucifer yellow^{(2 − )}, APTS ^{(3 − )}) and positively (Ethidium bromide⁽⁺⁾, DAPI⁽²⁺⁾ and propidium iodide⁽²⁺⁾) charged dyes. In all examples shown, the HelaCx43-EGFP cell pairs demonstrated at least one junctional plaque in the region of cell–cell contact. In all cases, the cell designated as cell 1 was patched with pipette containing dye. Images were recorded ~ 10 min after establishing a whole cell recording in cell 1. After allowing ~ 10 min for dye transfer, electrical cell–cell coupling was evaluated by establishing a whole-cell recording in cell 2 with the second pipette. All the cell pairs shown responded to heptanol (2 mM) treatment with uncoupling, which indicates that GJs and not cytoplasmic bridges were responsible for the dye transfer.

Fig. 7

Intercellular transfer of Alexa Fluor, a negatively charged dye, is restricted when channels are in the residual state. (A and B) Phase-contrast (A) and fluorescence (B) images showing a Novikoff cell pair in which dye transfer and electrical coupling were examined in open and residual states. Locations of pipettes 1 and 2 used for dual whole-cell recording and pipette 3 used for loading cell 1 with dye are indicated. RI-1, RI-2, and RI-3 are regions of interest from which fluorescence intensities were measured over time. Fluorescence intensities in the cells (RI-1 and RI-2) were calculated by subtracting the background fluorescence (RI-3). (C) Plot of normalized fluorescence intensity (FI) in cell 1 and cell 2 over time. A V_j of + 90 mV was imposed causing a decline in I_j to the residual conductance. Opening the patch in pipette 3 at steady state of g_j (see solid arrow) causes FI in cell 1 to rise. FI in cell 2 shows no change during the time when channels mainly reside in the residual state. Upon reopening channels by removal of V_j (see dashed arrow), FI begins to rise immediately in cell 2 and reaches ~ 14% of the maximum within 60 s. Imposition of V_j a second time caused an immediate decline in FI in cell 2 due to loss of transfer from cell 1 and dialysis with patch pipette 2. (D) Records of I_j and V₂ over time corresponding to fluorescence plot in (C). The V_j of + 90 mV imposed by stepping the voltage in cell 2 caused g_j to decline from 43 nS to a steady-state value of ~ 10 nS. Between the + 90-mV V_j steps, small repeated ± 10-mV V_j steps were applied to cell 2 to measure g_j. I_j recovered rapidly upon removal of the V_j step, indicating that mainly the fast gate was operating. Adapted from Ref. [62].

Fig. 8

Cx32-CFP and Cx43-EGFP form heterotypic junctions visible as large junctional plaques. (A) A phase-contrast image of HeLa cells expressing Cx43-EGFP (on top of white line) and Cx32-CFP (below white line). (B) A color overlay of fluorescence images of HeLaCx32-CFP shown in green and HeLaCx43-EGFP cells shown in red. The arrows indicate heterotypic junctional plaques (yellow regions).

Fig. 9

Asymmetric V_j gating in heterotypic Cx45/Cx43-EGFP junctions. (A) Pooled data of normalized steady-state g_j ( G_j) vs. V_j measured in 15 homotypic HeLaCx45 cell pairs. The solid line is a fit of all of the data points to a contingent gating model containing one gate per hemichannel with parameters A = 0.3 mV^{− 1} and V_o = 8.9 mV for negative V_j, and A = 0.17 mV^{− 1} and V_o = 7.5 mV [76]. The value of g_j reaches a minimum at ± 60 mV and then increases at higher V_js (inset; the solid line is a regression line of the first order). (B) Normalized g_j–V_j relation of a Cx45/Cx43-EGFP heterotypic junction. Data were pooled from 14 cell pairs. The thin black line is a fit of all data points to a four-state contingent gating model with one gate in each hemichannel (open and dotted circles). The parameters are A = 0.18 mV^{− 1} and V_o = 1 mV for the Cx45 hemichannel and A = 0.03 mV^{− 1} and V_o = 26 mV for the Cx43-EGFP hemichannel. The thick gray line is a fit of the data points indicated by dotted circles to 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 V_o = 3 mV for the slow gate and A = 0.46 mV^{− 1} and V_o = 10 mV for the fast gate. An expanded view (inset) demonstrates that the contingent model can explain the secondary increase in g_j at large V_js [76]. (C) Example of opening of Cx45/Cx43-EGFP channels during a V_j step of − 60 mV applied to the Cx43-EGFP cell. Gating transitions are slow and between open and closed states. During a subsequent V_j step of + 60 mV, channels close completely with a short latency. (D) During V_j steps of 90 mV negative on Cx43-EGFP side, Cx45/Cx43-EGFP channels gate between the open state with a conductance of ~ 55 pS and the fully closed state. During positive V_j steps, the channel gates to the residual state (see arrow and dashed line) or to the closed state (second and third positive steps). (E) An example of asymmetric V_j gating in a Schwann/Fibroblast cell pair in response to V_j steps of ± 50 mV. Adapted from Ref. [76].

Fig. 10

Asymmetry of electrical coupling mediated by heterotypic Cx45/Cx43-EGFP junctions. (A) The HeLaCx43-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 HeLaCx45 cell (V₂) was current-clamped, and the steady-state holding potential was varied from ~ 5 mV (0–20 s) to ~ − 8 mV (20–62 s). Electrical coupling was much greater for negative pulses and coupling asymmetry increased by making the Cx45 side more negative. (B) Schematic of a cell pair; voltage steps 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 experiments in which the Cx43-EGFP or Cx45 cell was voltage-clamped and stepped, 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. Adapted from Ref. [76].

Fig. 11

V_m and chemical gating in C6/9 cell pairs. (A) Shown is an example of V_m gating. Both cells were clamped to the same V_m ( − 35 mV) and a test pulse of 15 mV was applied repeatedly (1 Hz) to cell 2 to assay I_j (upward deflections in current record of cell 1, I₁). A hyperpolarizing step of − 50 mV has no effect on I_j, while the depolarizing step of + 80 mV produces a reversible decrease in g_j down to ~ 0. (B) Summarized g_j–V_j plot (normalized) measured in eight cell pairs. The continuous curve is a fit of the data to the Boltzmann equation. (C) Example of V_m dependence of g_j at the single channel level. Shown are I_j records obtained by using the double whole cell patch clamp method. V_m was imposed by applying equal and simultaneous polarizations to both cells. V_j was held constant throughout at − 15 mV. At V_ms in cell 1 of − 60, − 35 and − 10 mV, the channel gates between γ_open, indicated by level O, and γ_res (dashed line, R). Depolarization from − 60 to − 10 mV decreases the relative time spent in γ_open. Further depolarization to + 15 mV causes the channel to close, indicated by level C. At + 60 mV the channel resides only in closed state. The transition to closed state has an apparently slow time course and is ascribed to V_m gating. Transitions between γ_open and γ_res are rapid and are ascribed to V_j gating. (D–E) Effect of heptanol on the g_j–V_m relationship. (D) Exposure to 1 mM heptanol leads to a gradual decline in I_j. A conditioning pulse from − 35 to − 85 mV increases I_j, while a conditioning pulse to − 10 mV led to complete uncoupling. (E) Time course of g_j–V_m dependencies measured at different time intervals of C6/36 cell pair treatment with heptanol. V_m gating was measured under control perfusion (open circles) and 2 – 3 (filled circles), 3 – 4 (filled squares) and 4 – 5 min (filled triangles) under heptanol perfusion. Washout from heptanol returns the g_j–V_m curve close to that obtained under control conditions. Adapted from [46,52].

Fig. 12

Rapid effect of pH-induced closure in hemichannels indicates direct action of H⁺ on connexins. (A) An inside-out patch containing a single Cx46 hemichannel was subjected to repeated applications of pH 6.0 (region within the rectangle). Membrane potential was held at − 30 mV; hemichannel openings are downward deflections in current. Summation of similar currents from >100 traces obtained from a total of five separate patches shows no measurable delay between pH 6.0 application and the decrease in current. (B) Ensemble currents show a second effect of pH that is sensitive to the duration of acidification. Ensemble currents from multiple patches were normalized to the average prior to acidification. Recovery from 1- and 2-s applications was nearly 100%, whereas recovery from 5-s applications was only ~ 80%. Longer exposures further reduced degree of recovery. The same phenomenon was observed in cell–cell channels. Adapted from Ref. [95].

Fig. 13

Schematics of a Cx43 gap junction channel containing fast (arrow with circle) and slow (arrow with square) gates. (A) V_j initiates gating mediated by both fast and slow gating mechanisms. (B) V_m and chemical uncouplers initiate gating mediated by the slow gating mechanism in both hemichannels. Gating transitions measured during the first channel opening during de novo channel formation are also slow and stepwise resembling gating transitions induced by V_m and chemical uncouplers.