Determinants of Cx43 Channel Gating and Permeation: The Amino Terminus

Abstract

Separate connexin domains partake in proposed gating mechanisms of gap junction channels. The amino-terminus (NT) domains, which contribute to voltage sensing, may line the channel’s cytoplasmic-facing funnel surface, stabilize the channel’s overall structure through interactions with the transmembrane domains and each other, and integrate to form a compound particle to gate the channel closed. Interactions of the carboxyl-terminus (CT) and cytoplasmic loop (CL) domains underlie voltage- and low pH-triggered channel closure. To elucidate potential cooperation of these gating mechanisms, we replaced the Cx43NT with the Cx37NT (chimera Cx43^∗NT37), leaving the remainder of the Cx43 sequence, including the CT and CL, unchanged. Compared to wild-type Cx43 (Cx43WT), Cx43^∗NT37 junctions exhibited several functional alterations: extreme resistance to halothane- and acidification-induced uncoupling, absence of voltage-dependent fast inactivation, longer channel open times, larger unitary channel conductances, low junctional dye permeability/permselectivity, and an overall cation selectivity more typical of Cx37WT than Cx43WT junctions. Together, these results suggest a cohesive model of channel function wherein: 1) channel conductance and size selectivity are largely determined by pore diameter, whereas charge selectivity results from the NT domains, and 2) transition between fully open and (multiple) closed states involves global changes in structure of the pore-forming domains transduced by interactions of the pore-forming domains with either the NT, CT, or both, with the NT domains forming the gate of the completely closed channel.

Introduction

Connexins and the channels they form, gap junction channels (GJChs) for intercellular signaling and hemichannels (HChs) for transmembrane (TM) signaling, facilitate coordinated tissue function in a connexin (Cx) and tissue-specific manner (1). The conserved membrane topology of the >20 members of the Cx gene family includes four TM domains (TM1–TM4), two extracellular loops (ECL1 and ECL2), and the intracellular cytoplasmic loop (CL), amino-terminus (NT), and carboxyl-terminus (CT) (1). TM1–TM4 and ECL1–ECL2 comprise what we refer to as the pore-forming domains (PFD). HChs are formed by six Cxs arranged as a torus with axis perpendicular to the (single) plasma membrane; GJChs are formed by the extracellular, coaxial docking of two HChs from neighboring cells.

The extent and character of intercellular signaling supported by GJChs depend on the number of channels (N), their individual permeability to current carrying ions (unitary conductance; γ_j) and other small molecules (p_j), and their probability of being open (P_o) (2, 3). Numerous studies of these determinants of intercellular signaling for many members of the gene family, expressed in isolation or together with other family members, have been performed with a primary goal of determining which Cx domains and specific amino acids therein, are responsible for specific properties of the channel and protein, e.g., channel gating and permselectivity, cell growth regulation, and channel assembly (3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

Complete or partial closure of GJChs can be triggered by voltage (transjunctional and/or TM), low intracellular pH, elevated Ca²⁺, volatile anesthetics, medium chain length alcohols, unsaturated fatty acids, and Cx phosphorylation (1, 13, 14, 15). This multiplicity of gating effectors and the relatively few channel domains available to mechanistically support gating raise important questions, for instance, on specificity of gating mechanism (domain) across Cx family members, tissue specificity of gating for any given Cx and trigger, and impact of coexpression of multiple Cxs able to form mixed GJChs (and HChs). Chimeras of two Cxs with interchanged domains (5, 16, 17) and Cxs with truncations (7, 8, 16, 17), deletions (6), and site mutations (15, 18, 19, 20, 21, 22) have been very useful in discerning roles of specific domains and residues in gating of GJChs. These structure-function approaches and high resolution structural studies have led to two distinct models for channel closure: 1) Coincident radial expansion of the cytoplasmic aspect of the TM domains and movement of the NTs toward the cytosol to form a gate (plug) to the GJCh, the NT-plug model ((23, 24, 25, 26, 27, 28); cf. (10, 29, 30)), and 2) interaction of the CT with the CL to close or partially close the GJCh, the Particle-Receptor model ((2, 4, 6, 7, 8, 31); cf. (32, 33)). A previously advanced Global structure destabilization model (34, 35, 36, 37), wherein gating is triggered by changes in membrane fluidity, is compatible with either the NTs or CTs forming the actual gate. However, whether these mechanisms are mutually exclusive or cooperate in a complex regulation of channel gating remains uncertain. Here, we explore this possibility by asking whether changes in one model constituent, the NT, affect the function of the ostensibly separate particle-receptor interaction.

Members of the Cx gene family have unique patterns of expression that frequently include coexpression of two or more isotypes in a cell type (38). Although Cx isotypes share significant sequence similarity, their differences, even among the most similar isotypes, underlie critical functional differences (39, 40, 41, 42, 43, 44). Cx43 and Cx37 coexpress in several cell types in vivo including vascular endothelial and smooth muscle cells, where the functional consequences of their expression can be quite different (45, 46). In vitro, these Cxs are able to form functional heteromeric channels with variable permeability and gating features (47, 48). We previously examined the conductance, charge selectivity, and permeation properties of Cx43, Cx37, and Cx43^∗CT37 (chimera of Cx43 with the CT of Cx37) channels and junctions, and showed that Cx43^∗CT37 channels (like the CT-truncated Cx43 mutant, M257 (8)) rarely close to a subconductance state, existing instead in either a fully open state of similar γ_j to Cx43 wild-type (Cx43WT) or a closed state (17). Furthermore, although Cx43^∗CT37 displays charge selectivity comparable to Cx43WT, the permselectivity of Cx43^∗CT37 junctions is nearly 10-fold lower than that of Cx43WT junctions, suggesting that the CT of Cx43 must interact with the PFD to modulate channel permeability and permselectivity to levels greater than are intrinsic to the native PFD (17). Because the NT of at least some Cxs is thought to harbor channel selectivity, permeability, and gating determinants (10, 13, 22, 49, 50, 51, 52), in this study we examined these parameters of channel function in a Cx43^∗NT37 chimera, wherein the first 22 residues of Cx43 (MGDWSALGKLLDKVQAYSTAGG) were replaced with those of Cx37 (MGDWGFLEKLLDQVQEHSTVVG). We show that this chimera formed GJChs extremely resistant to many known triggers of channel closure, and that channel γ_j values were larger than expected for Cx43WT despite significant cation selectivity and more restrictive size selectivity. Our results provide new, to our knowledge, insights into channel gating mechanisms and the regulation of junctional coupling.

Materials and Methods

Plasmid construction

The chimera Cx43^∗NT37 was constructed from two polymerase chain reaction (PCR) fragments designed to replace the Cx43NT (r/mCx43: MGDWSALGKLLDKVQAYSTAGG) with the Cx37NT (mCx37: MGDWG FLEKLLDQVQEHSTVVG). The first fragment was made by amplifying the sequence encoding amino acid residues 1–23 (residue 23 and its codon are common to both sequences) of Cx37 using the 5′ forward primer [Primer I] from the pTRE2 vector sequence (5′-CGCCTGGAGACGCCATCC-3′) and a 3′ reverse primer [Primer II] with sequence identical to mCx43 and mCx37 (5′-GAGCACTGACAGCCACACCTTGCCCACCACG-3′). The second fragment was made by amplifying the sequence encoding amino acid residues 24–382 of mCx43 also from the pTRE2 vector, using a 5′ forward primer [Primer III] whose sequence is complementary to Primer II (5′-CTCGACCGTGGTGGGCAAGGTGTGGCTGTCAG-3′) with additional 5′ mCx37 sequence and the 3′ reverse primer [Primer IV] from the pTRE2 vector (5′-CTCCCGGCTGACGCCCT TGAAGAAGACATA-3′). These two PCR products were purified and subsequently used in a PCR reaction with Primer I and Primer IV to yield the full chimera sequence. The amplified product was digested with BamHI/NotI and ligated into the pTRE2hygro vector to generate pTRE2h-Cx43^∗NT37. Sequence was confirmed at the University of Arizona UAGC Sequencing Facility. Similar approaches were used to make the opposite chimera, Cx37^∗NT43, but this protein did not traffic properly and did not form functional channels.

Cell culture and transfections

iRin cells, previously described (53), were transfected with Lipofectamine 2000 (Life Technologies, Grand Island, NY) and the pTRE2h-Cx43^∗NT37 plasmid. Subclones were isolated by dilution cloning and tested for gene expression by Western blotting. For selected subclones, the dose and time-dependent response to doxycycline induction of gene expression was determined and expression quantified as previously described (54, 55). Subclones or recently transfected cells were examined. Control experiments were performed in rat insulinoma (Rin) cells stably transfected with Cx43 (Rin43) (56).

Immunofluorescence

Staining was performed as previously described (53); briefly, cells plated on glass coverslips, were fixed, triton-treated, and their nonspecific reactivity blocked before incubation with primary antibody against Cx43 (Sigma-Aldrich, St. Louis, MO) and secondary Cy3 conjugated antibody (Jackson Immunoresearch, West Grove, PA).

Electrophysiology

Electrical recordings were performed as previously described (3) using dual or single whole-cell clamp protocols (pClamp software, Molecular Devices, Sunnyvale, CA), Axopatch2 (Molecular Devices), or SEC-05LX amplifiers (NPI Electronic GmbH, Tamm, Germany), and osmotically matched external and pipette solutions. Macroscopic conductance (g_j) was explored with transjunctional voltage (V_j) pulses of ±10 mV and V_j-gating assessed with square 5 s voltage pulses of increasing magnitude from 0 to ±100 mV in 10 mV (step protocol). V_j-dependence and fast inactivation were analyzed with Origin software (OriginLab, Northampton, MA). Open state stability was measured with V_j = ± 40 or 80 mV in poorly coupled cell pairs (see the Supporting Material for details on measurement of P_o). Channel conductance (γ_j) was measured with V_j = ± 40 or 80 mV in these poorly coupled cell pairs or in pairs whose initial g_j was reduced with halothane. For halothane-induced uncoupling, a 1–2 ml bolus of halothane solution was added to the recording chamber (total volume of ∼3 ml) in the immediate vicinity of the studied cell pair; successive exposures to increasing concentrations were thus possible. Different halothane concentrations were obtained by diluting a stock of saturated halothane (∼17 mM at 20°C (35); in external solution) with halothane-free external solution (i.e., 1:4, 1:3, 1:2 v/v). All-points histograms were produced from short (5–12 s) fragments of extended recordings to accurately identify and illustrate channel open states. Clear channel transitions with current levels enduring ≥50 ms were measured to build event histograms, as customary (3, 57). Low pH-induced uncoupling was achieved by continuously superfusing cells with bicarbonate-containing solution adjusted to pH = 6.0 to 6.4 (bubbling with 95% CO₂/5% O₂.). Only experiments with high resistance seals (GΩ values) and good intercellular access (wherein junctional current (I_j) in the nonpulsed cell and total current in the pulsed cell (I_T: sum of membrane (I_m), leak and I_j currents) were of similar amplitude but opposite polarity) were analyzed.

Dye injection

For quantitative dual-dye coupling experiments, cationic NBD-m-TMA (NBD), anionic Alexa350 (Alx350), and rhodamine-labeled dextran 3000 Da (rhodex3000) were injected into one cell of a pair in a single bolus, and their diffusion to the neighboring cell documented for 10–20 min with a fluorescence microscope, as previously described (3). Transjunctional dye diffusion was quantified as decreasing fluorescence in the donor and simultaneous increasing fluorescence in the recipient cell over time (58, 59). To assess permselectivity, NBD and rhodex3000 were delivered through a patch pipette in whole cell mode. In this circumstance, fluorescence increases toward plateau levels in the donor and recipient cells, with fluorescence in the recipient cell lagging the donor in a manner directly related to the coupling level (3, 9). For each junction and dye, a diffusion rate constant (k), as index of the junctional permeability to the dye (P_j-dye), was calculated and plotted against that of the coinjected dye (charge selectivity, P_j-NBD/P_j-Alx350) (58, 59) or against the associated g_j level (permselectivity, P_j-NBD/g_j (3, 9)).

Statistical analysis

Values are reported as mean ± SE. Where needed, comparisons were performed by t-test (3, 57), with significance at p < 0.05.

Results

Cx43^∗NT37 channels are resistant to gating by halothane, low pH, and voltage

iRin43^∗NT37 cells expressed Cx43^∗NT37 abundantly, with protein detected intracellularly as well as in appositional membranes (Fig. 1 A). Cell pairs were frequently well coupled and not readily uncoupled by ∼5.6 mM halothane, a reproducible feature of Cx43WT channels and Rin43 cells (3, 9, 35). Only small and/or transient decreases in coupling were seen upon application of 4.25–8.5 mM halothane, as illustrated in Fig. 1, B and C. To assess whether this halothane resistance was due to an abnormally high incidence of cytoplasmic bridges in iRin43^∗NT37 cells, the probe rhodex3000 was included in one electrode during measurements of g_j. No intercellular diffusion of rhodex3000 was observed in well-coupled pairs resistant to halothane-induced uncoupling (n = 4). A halothane-resistant cell pair exposed to successive bolus infusions of increasing concentrations of halothane is shown in Fig. 1 C. Only 17 mM induced sustained, but still reversible (not shown) uncoupling. After partial uncoupling with 4.25–8.5 mM halothane, when junctional conductance was low (≤1 nS), GJCh activity (mirrored in both current traces) with abundant flickering was observed, but clear and stable channel events were not easily recognized (Fig. 1 D). That Cx43^∗NT37 junctional channels showed low susceptibility to halothane-induced uncoupling indicated a substantial alteration of the channel closure mechanism(s) and raised the possibility that closure in response to other gating agents might also be compromised.

Figure 1.

Cx43^∗NT37 gap junctions are halothane resistant. (A) Immunofluorescence of Cx43^∗NT37 protein (white stain) demonstrating high expression levels in doxycycline-induced (left) versus noninduced (right) cell; inset: ∼2-fold magnification of likely junctional plaque (red arrow). (B) Successive total (I_T) and junctional (I_j) current traces (pulses start every 10 s) from a coupled cell pair showing transient electrical uncoupling induced by halothane (∼5.6 mM); notice that after complete (^∗) uncoupling, full recovery of junctional current occurs within 10 s (#) in the absence of halothane washout. (C) Multiple partial and transient junctional uncoupling episodes caused by increasing concentrations of halothane (in mM: ^∗5.6, ^∗∗8.5, and ^∗∗∗17) demonstrating low susceptibility of the Cx43^∗NT37 mutant junctions to the anesthetic; inset: DIC and fluorescence images of this cell pair showing junctional impermeant rhodex3000 (red, false color) stays in the injected cell, thus confirming the presence of a gap junction. (D) Closer view of I_T and I_j currents during spontaneous recovery from halothane-induced uncoupling (^∗; from an out of scale, initial g_j value of ∼12 nS) revealed the archetypal mirror image of gap junctional recordings. To see this figure in color, go online.

Intracellular acidification, by continuous superfusion of CO₂-saturated solutions (buffered to pH 6.0), caused complete uncoupling of Cx43WT expressing cells within 5 min, but in Cx43^∗NT37 expressing cells >50% of initial conductance (53.6 ± 0.1%, n = 3) remained after 15 min (Fig. 2 A, Cx43^∗NT37). This low susceptibility to acidification-induced uncoupling indicated a failure of the low pH-gating mechanism, a property of Cx43WT generally attributed to CT-CL interaction (60). Similarly, V_j-gating of Cx43^∗NT37 junctions also differed from Cx43WT junctions. Fast inactivation was confirmed for Cx43WT, but was not observed in Cx43^∗NT37 junctions of comparable g_j values (Fig. 2, B and C). A slow decrease of junctional current (slow gating?) was occasionally observed at high V_j ≥ 80 mV in Cx43^∗NT37 junctions (Fig. 2 C, some low G_j values at higher V_j gradients). Because fast inactivation of Cx43 channels is also attributed to CT-CL interaction (8, 20, 61), our results suggested this interaction was absent or its effects were critically impaired in Cx43^∗NT37 junctional channels.

Figure 2.

Compromised pH- and V_j-gating of Cx43^∗NT37 gap junctions. (A) Effect of low pH on Cx43WT (n = 4; initial g_j values: 8.0, 20.2, 41.2, and 11.3 nS) and Cx43^∗NT37 (n = 3; initial g_j values: 28.4, 15.3, and 12.0 nS) gap junctions; g_j was recorded while cells were superfused with acidifying solution (see Materials and Methods; external pH measured at superfusion time was 6.4, 6.0, 6.0, 6.4 for Cx43 and 6.0 for all Cx43^∗NT37 junctions); for each cell pair, the first g_j value (time zero) is the average of the minute previous to the superfusion start, and all values were normalized to the highest g_j for the rest of the experiment; in all, rhodex3000 was used (as in Fig. 1C) to recognize bona fide gap junctions (not shown). (B) Illustrative junctional currents elicited by a high amplitude V_j pulse on Cx43WT- and Cx43^∗NT37-expressing iRin cells; V_j-induced fast I_j inactivation was seen in Cx43WT (white dashed line: fit to a second order exponential function, tau and initial g_j values displayed), but not in Cx43^∗NT37 junctions. (C) V_j-dependence of Cx43WT and Cx43^∗NT37; I_j traces elicited by a step protocol (see Materials and Methods) were measured and steady-state g_j (g_j^ss; at 5 s) was divided by its corresponding instantaneous g_j (g_j^inst; first milliseconds) to obtain normalized G_j (g_j^ss/g_j^inst) values, which were then used in a Boltzman equation fitting. For Cx43 (g_j = 2.93 ± 0.28 nS, n = 3), V₀ = 69.65 mV (R² = 0.97), and −68.40 mV (R² = 0.93). The Cx43^∗NT37 data (g_j = 2.80 ± 1.02 nS, n = 9) could not be fit with a Boltzmann equation.

Cx43^∗NT37 channels—open state stability and conductance

The macroscopic data in Figs. 1 and 2 suggested that the P_o of Cx43^∗NT37 channels was unusually high and resistant to reduction by standard triggers of gating. Multiple channels are typically active in coupled cell pairs making it difficult to assess P_o and γ_j; however, in several of our cell pairs, the level of coupling was 200 pS or less without application of halothane. We examined these cell pairs for single-channel events and open state stability (Fig. 3, A and B).

Figure 3.

Cx43^∗NT37 channels are more conductive than Cx43WT channels and transition to a residual state less frequently. Cx43WT (A) and Cx43^∗NT37 (B) channel activity at V_j = 40 and 80 mV, with corresponding all points histograms to the right of each trace; V_j pulses (as applied to opposite side) depicted above each trace; long-dash lines indicate baseline, short-dash lines mark open state current levels, numbers denote full open state conductances; red symbols mark spontaneous opening (^∗) and closing (#) current transitions not associated with V_j change. (C and D) Histograms of event amplitudes of Cx43WT and Cx43^∗NT37. The novel (left and right) graphical display facilitates comparison of conductance along the ordinate axis and the difference plots highlight the subpopulations of channels more abundant at each V_j. n, experiments; N, transitions. At V_j = 40 mV, Cx43WT (C) displays multiple channel amplitudes with a major peak at ∼72 ± 0.3 pS (an intermediate value), whereas, at V_j = 80 mV, transitions of 31 ± 3.5 pS and 112 ± 1.5 pS are more evident. In comparison, Cx43^∗NT37 (D) at V_j = 40 mV displays a high incidence of 109 ± 8 pS and larger transitions, whereas at V_j = 80 mV intermediate values (72 ± 5 pS) are more abundant. Transitions between residual and closed states were less frequent in the Cx43^∗NT37 mutant than in Cx43WT. To see this figure in color, go online.

Two poorly coupled Cx43WT expressing cell pairs were identified, one with a macroscopic junctional conductance (g_j) close to zero (Fig. 3 A), the other with a g_j near ∼200 pS (Fig. S1). Three poorly coupled Cx43^∗NT37 expressing cell pairs were identified, each with a g_j approximating 125–150 pS (representative pair illustrated in Fig. 3 B). P_o, cumulative pulse time, average open time, and time to closure following pulse onset are summarized in Table 1. The open probability of the Cx43^∗NT37 channel was considerably larger than for Cx43WT at both V_j (40 and 80 mV) values, as were the mean open time and time to closure following pulse onset. Fig. S2 summarizes the open duration as a function of pulse length for all pulses; the graph suggests that the open time average for Cx43^∗NT37 channels is likely underestimated as a number of pulses were insufficiently long for closure to occur. Similar results were obtained in two additional Cx43^∗NT37 cell pairs. The Cx43^∗NT37 results stand in stark contrast to the behavior of Cx43WT and are consistent with the results in Figs. 1 and 2 showing resistance of Cx43^∗NT37 macroscopic conductance to decrease when challenged with standard triggers of gate closure. These data strongly suggest that the stability of the open state of Cx43^∗NT37 GJCh far exceeds that of Cx43WT.

Table 1.

Channel Properties of Cx43WT and Cx43^∗NT37: No Halothane

	Cx43WT	Cx43∗NT37
±40 mV
Po	0.126	1.0
Pulse time (s)	118	79
Open time (s)	2.96 ± 1.16	9.86 ± 0.77
Time to close (s)	1.94 ± 0.0	NA
±80 mV
Po (s)	0.015	0.252
Pulse time (s)	253	283
Open time (s)	0.43 ± 0.12	0.97 ± 0.14
Time to close (s)	0.14 ± 0.06	0.86 ± 0.12
Conductance (pS)	117 ± 2.9	140 ± 4.0

In the absence of gap junction blockers, the open probability and open time of the Cx43^∗NT37 are higher than those of Cx43WT at two different V_j values; in addition, the Cx43^∗NT37 channels open to a larger conductance and are less V_j sensitive.

Because the macroscopic conductance of most cell pairs exceeded 1 nS (approximating 10 simultaneously open channels), halothane was used to reduce P_o such that individual channel events could be observed. Interestingly, in the presence of halothane the flickering activity observed in poorly coupled Cx43WT expressing cells decreased as the activity of individual channels became evident (Fig. S1 A). In contrast, flickering activity was increased by halothane in Cx43^∗NT37 expressing cells (Figs. 1 and S1 B). Furthermore, the P_o of Cx43^∗NT37 channels was additively/synergistically reduced by the action of V_j and halothane, as evident from the induction of spontaneous opening and closing events at V_j ± 40 and 80 mV by halothane (Figs. S1, S3, and S4).

The unitary conductance of Cx43WT and Cx43^∗NT37 expressing cells was measured from naturally occurring and halothane-induced poorly coupled cell pairs. All points histograms from segments of traces suggested the fully open Cx43^∗NT37 channel was more conductive than Cx43WT channels (∼140 pS vs. ∼110 pS; Fig. 3, A and B; Table 1). Transitions between conductive and closed states (at V_j = ±40 mV and ±80 mV) for Cx43WT (Fig. 3 C) ranged from 30 to 120 pS. The difference plot suggests that at ±40 mV the WT channel transitioned between the fully open and residual state; these transitions also occurred at ±80 mV, but transitions to the closed state and from the residual to closed state were more common than at ±40 mV. For Cx43^∗NT37 (Fig. 3 D) transitions ranged from 20 to ∼160 pS. At ±40 mV the difference plot suggests the chimera transitioned between fully open and a residual state, but the channel rarely closed completely from the residual state, and thus an event of this amplitude was not observed as a distinct peak in the histogram. At ±80 mV, it appears that the chimera transitions between unique (relative to ±40 mV) subconductance states, perhaps reflecting the combined effects of halothane and large V_j (cf. Figs. S1, S3, and S4).

Cx43^∗NT37 junctions are charge and size selective

Unlike Cx43WT, Cx43^∗NT37 junctions were cation selective. Cx43WT junctions are not charge selective for current carrying or larger ions (with limiting diameters of ∼5 Å (58, 59)). This lack of charge selectivity is not modulated by the CT, as its removal, presence in excess, phosphorylation (59), or replacement with the CT of Cx37 (Cx43^∗CT37 (17) and Fig. 4) fail to alter Cx43 GJCh charge selectivity. In contrast, Cx43^∗NT37 junctions exhibited an approximately sixfold preference for NBD⁺ over Alexa350⁻, comparable to the charge preference of Cx37WT junctions (Fig. 4). This result suggests that the NT determines, at least in part, the charge selectivity of GJChs, consistent with the observations of others (see (51)).

Figure 4.

Charge selectivity of Cx43^∗NT37 junctions. (A) DIC and fluorescence (false color) images illustrating transjunctional diffusion of NBD and Alexa350 (but not rhodex3000) between paired cells expressing Cx43^∗CT37 (left) and Cx43^∗NT37 (right); displayed time (minutes) apply to each row of images. Notice that both dyes diffuse well in the Cx43^∗CT37 pair, but diffusion of Alexa350 is slower than NBD in the Cx43^∗NT37 pair. Cal: 10 μm. (B) Analysis of the concurrent dye diffusion of the cells in (A); notice that k_NBD and k_Alexa350 are similar for the Cx43^∗CT37, but differ by almost an order of magnitude for the Cx43^∗NT37 mutant. (C) Plot of k values obtained from the two dyes, for the parental and mutant connexins indicated; notice that the slope of Cx43^∗NT37 pairs (C, top, n = 8) is similar to that of Cx37 (C, bottom, n = 10), and that both display overall low permeability. In contrast, the slope of Cx43^∗CT37 (n = 8) resembles that of Cx43WT (n = 25; at this scale, chosen to better display the slope of the low permeability junctions, not all Cx43WT data points are shown). (D) Comparison of the charge selectivity of the indicated parental and mutant connexins; the cation selectivity of Cx43^∗NT37 resembles Cx37. To see this figure in color, go online.

Cx43WT gap junctions display a broad size selectivity that manifests as variable permselectivity (dye versus current) and is regulated in a CT-dependent manner (9). Because only the NT sequences differ between Cx43^∗NT37 and Cx43WT, we next compared, quantitatively, the NBD permselectivity of Cx43^∗NT37 junctions to Cx43WT and Cx37WT junctions, and for completion, to Cx43^∗CT37. Cx43^∗NT37 junctions showed very low NBD permeability and much lower NBD permselectivity than Cx43^∗CT37 and Cx43WT (Fig. 5). Notice that for g_j values in the same range (see Fig. 5 C), Cx43^∗NT37 junctions displayed permeation rate constants at least one order of magnitude lower than those of Cx43WT junctions, and comparable to Cx37WT junctions. These data indicate that despite their enduring residence in the fully open conductance state, Cx43^∗NT37 GJChs were far more size restrictive than Cx43WT GJChs (see Fig. 5 D), instead resembling the size selectivity of Cx37WT GJChs (3).

Figure 5.

Permselectivity of Cx43^∗NT37 junctions. (A) DIC and fluorescence (false color) images illustrating transjunctional diffusion of NBD (but not rhodex3000) between paired cells expressing Cx43^∗NT37; Cal: 10 μm; displayed time in minutes. (B) Analysis of dye coupling to obtain a diffusion rate constant (k) and electrical coupling (inset) for the pair in (A). (C) Plot of k values versus their corresponding g_j values, for the parental and mutant connexins named in (D); notice that the Cx43^∗NT37 pairs (n = 5) display a slope comparable to Cx37 (n = 14) and that both Cx37 and Cx43^∗NT37 have an overall low permeability. Cx43WT (n = 54) and Cx43^∗CT37 (n = 28) data is a compilation of published and unpublished experiments, of which only those with values within the displayed conductance range are displayed, for proper comparison. (D) All points and box plots of the NBD permselectivity values of the indicated parental and mutant connexins (Cx43: 0.658 ± 0.087; Cx43^∗CT37: 0.022 ± 0.0022; Cx37: 0.008 ± 0.002; Cx43^∗NT37: 0.008 ± 0.002); the permselectivity of Cx43^∗NT37 resembles Cx37.

Discussion

Based on differences in function of homomeric-homotypic Cx37WT and Cx43WT channels, heteromeric Cx43-Cx37 channels (47, 48), and the assumption of independent gating by the NT and CT domains, we expected Cx43^∗NT37 channels would gate rapidly to a residual subconductance state with Cx37-like V_j sensitivity, close fully in response to cytosolic acidification or halothane, display Cx37-like cation selectivity, but display Cx43-like size selectivity. Instead, Cx43^∗NT37 channels were stably open, resistant to partial or complete closure by V_j, acidification or halothane, and, although cation selective, the permselectivity of these channels was extremely low (Fig. 6). These observations offer new, to our knowledge, insights on the roles of the NT, PFD, and CT in channel gating and permeation, as discussed below.

Figure 6.

Summary of channel behavior findings for parental Cx43, Cx37, and Cx43 mutants. Comparison of the NT sequence of Cx43 and Cx37 shows a more negatively charged Cx37NT. Color-coded comparison of gating and selectivity indicates that the mutant Cx43^∗NT37 channel behavior is not entirely predicted by that of the parental proteins; instead, Cx43^∗NT37 channels display features suggestive of a CT-CL interaction that is weak (as in the chimera Cx43^∗CT37) or absent (as in the truncated Cx43tr257 mutant), and permselectivity/charge selectivity resembling that of Cx37. n.a., not available. To see this figure in color, go online.

Gating

The resistance of Cx43^∗NT37 GJChs to gating by all tested triggers implies a higher energy barrier must be crossed for the chimeric channel to move from the open state to either the partially closed or closed states. To understand how replacing the NT of Cx43 with the NT of Cx37 could alter the gating behavior of the channel so drastically, it is helpful to look for common elements in current models of GJCh gating (see the thorough review of Bargiello and colleagues (13)).

The NT-plug model postulates that the NTs fold back toward the pore to line the vestibule of the channel where they stabilize its open state through hydrophobic interactions of the helical NT with the helical TM1 (W3 to M34) and through formation of an interprotomer ring of hydrogen bonds involving D2 and T5 of each subunit. The stabilizing effects of the NT are postulated to be overcome by triggers of gating (voltage, lipophile) that cause radial expansion of the TM domains and movement of the NTs toward the cytosol where, together, they form a hexameric NT plug (closed gate conformation). This model derives its support from elegant high-resolution structure studies of Cx26-M34A (23, 26) and Cx26WT (24). Molecular dynamics (MD) simulations suggest this crystal structure represents the closed rather than open state of the channel; the open conformation suggested by MD has the same overall structural features as the crystal structure, but the packing of the four TM helices is relaxed and their tilt angle differs such that pore diameter in the region of the NT is increased (62) and similar throughout the length of the open pore (Fig. 7). Addition of energy (e.g., temperature, voltage, lipophile (through membrane fluidity) (13)) is predicted to destabilize open-state interactions, enabling channel closure (Open → Substate ^∗ → Closed in Fig. 7). Comparable data are not available for Cx43. In addition, it is less clear in the NT-plug model how altered phosphorylation of the CT (which does not occur in Cx26 but certainly occurs in Cx43) or low pH would close the channel (but see (63) for a plausible, NT-based scenario for Cx26).

Figure 7.

Potential unified model of Cx43WT and Cx43^∗NT37 channel gating. Cx43WT: Closure to a subconductance state can be triggered by changed interaction of either the NT (substate ^∗, top) or CT (substate ▾, bottom) with the PFD. (For clarity, the altered conformation is shown only for the right half of the channel, and only one CT is included). Note narrowing of the pore occurs in both, despite radial expansion of the vestibule induced only by the NT. CT conformational change shown as a change in shape of the CT particle. Whether the CT is bound in the fully open channel is unknown, hence the two possible positions for the CT. The closed state involves changes in interaction of both the CT and NT. Cx43^∗NT37: Because the Cx37NT is unkinked, it is unable to fold into the vestibule and interact with the PFD. The pore diameter is stably open and pore length reduced. The CT can still interact with the PFD to partially close the channel to a substate conductance, but is less likely to do this with any known gating trigger. To see this figure in color, go online.

The CT-Particle-Receptor model proposes that the CT, acting as a particle, partially or completely plugs the fully open channel by binding to the channel’s receptor ((8, 31, 60, 64); cf. (32, 65, 66, 67)). This model derives support principally from functional data on Cx43 and Cx40 (wherein channel closure is induced by transjunctional voltage, low pH, or CT phosphorylation changes), but also from NMR structural data. Partial versus complete GJCh closure with different gating triggers suggests multiple possible conformations of particle-receptor interaction, likely reflecting different conformations of the receptor, the particle, or both (Open → Substate ▾→ Closed in Fig. 7). Large V_j differences are envisioned to alter the receptor’s structure to enhance the probability of particle binding and consequent stabilization of a partially closed state (referred to as the residual state) the conductance of which reflects a narrower channel diameter and/or longer channel length (8, 61); pH reduction is envisioned to alter the structure of both the receptor and particle (31, 68, 69), increasing their mutual affinity, with interaction resulting in complete channel closure (70); phosphorylation is envisioned to alter the structure of the particle (71) thereby increasing or decreasing its tendency to bind to the receptor, possibly regulating channel P_o and/or conductance. The elements of the channel comprising the receptor’s binding pocket remain unclear, although the distal region of the CL (L2) is apparently involved (68, 69, 72, 73); the portions of the CT able to interact with the receptor binding pocket also remain unclear, although the last 5–10 residues appear to interact (6, 74, 75, 76). Of importance, it remains uncertain whether particle-receptor interaction represents gate formation, or if this interaction induces a global change in channel structure that enables formation of the gate by another domain (possibly by the NT).

Both the NT-plug and Particle-Receptor models are compatible with gating induced by lipophilic agents, as these agents are thought to increase membrane fluidity and thereby destabilize the open state. Lipophile-mediated gating models derive their support from functional studies showing reversible loss of coupling in a dose-dependent manner to many lipophilic compounds. For Cx43 channels, the lipophilic compounds that induce closure all increase the fluidity of the internal region (C9–C18) of the membrane bilayer (34, 35, 36), suggesting a destabilization of the channel’s global structure in this region (37). Whether the NTs or CTs form the actual gate or a region in the middle (z axis) of the channel forms the gate (slow gate?) is unclear. Of importance, every gap junction channel studied to date, irrespective of connexin composition, can be closed by lipophilic agents.

It is important to realize that these models of gating are not mutually exclusive (as suggested previously by Maeda and colleagues (24)). The gating behavior of Cx43^∗NT37 suggests an integrated model wherein the NT has global effects on all three types of gating. The intent of the model, presented next, is to show how existing models of gating can be integrated to explain Cx43^∗NT37 behavior, thereby generating testable hypotheses to understand gating.

Toward an integrated model of connexin channel gating

The similar topologies of all connexins and gating behaviors of all Cx-comprised GJChs suggest that the interactions underlying each of the gating models apply to all connexins and work together (on the channel’s structure) to modulate gating, as suggested by the comparative analysis of the crystal structure, MD simulations, and functional data (13). In Fig. 7, we attempt to encapsulate an integrated model of GJCh gating (potentially appropriate to all connexins) wherein channel closure involves a change in structure of the PFD (radial expansion at the pore’s orifice, constriction in the pore’s middle) that can be triggered by agents that alter the interactions of the PFD with the CT, NT, or lipid bilayer and are permissive to gate formation, likely by the NT (23, 24). Triggers of altered interactions are expected to include, lipophiles (e.g., halothane and octanol), V_j, low pH, and CT phosphorylation. Before discussing how our results led us to this view of channel gating, let us first address the question of how differences in the dynamic structure of the Cx43 vs. Cx37 NTs might produce a stably open channel.

Structure (NMR)-function studies of the NTs of WT and mutant forms of Cxs 26, 37, and 43 reveal interesting differences between them. For Cx26, Purnick and colleagues (30) showed that the first 10 NT residues form an α-helix that, due to a glycine hinge at residue 12, could fold back toward the pore to form the surface of the funnel-shaped channel entrance, a possibility consistent with two-dimensional and three-dimensional structural studies of this connexin (23, 24). Mutations at Cx26-G12 that preserve a kinked NT structure (proline) preserve function of Cx26 channels, whereas mutations that tend to extend the helical region (S, Y, V), thereby reducing hinge function, render the channel nonfunctional. Similarly, Shao and colleagues (52) showed that the WT Cx43NT is kinked and mutations that eliminate the kink render the channel nonfunctional. In contrast to these connexins, Kyle and colleagues showed that the Cx37NT does not have a comparably placed hinge point. Instead its structure is disordered before and possibly after a helical region formed by residues 5 through 16 (50). Mutations that disrupt this region of helical structure, proline substitution at positions 10 and 15, render the channel nonfunctional (likely stabilized in the closed state, because trafficking of the mutant protein to GJ plaques is not prevented). This difference in structure between the Cx37NT versus Cx26 and Cx43 NTs suggests possible differences in the function of the NT in homomeric channels of these connexins. Perhaps in Cx37 channels, the NTs do not fold back into the pore’s orifice (thereby contributing to the large conductance of this GJCh) but nonetheless are able to form the gate responsible for closing the channel. Regardless, it appears that the Cx37NT may be unable to line the vestibule of the Cx43 channel (chimera in Fig. 7) and that the extremely stable open state of the Cx43^∗NT37 channel reflects loss of NT-PFD interactions and gain of other interactions within and between protomers that serve to stabilize the open state of the channel. Consistent with this possibility, the unitary conductance of Cx43^∗NT37 channels was larger than that of Cx43WT, possibly reflecting a larger vestibule diameter (no NT lining) and/or shorter length of the reduced-diameter pore in the chimera (chimera in Fig. 7). Clearly, more studies will be necessary to resolve these possibilities, but it is an intriguing idea that the open-state structure of a gap junction channel could be more stable without NT-PFD interactions than with those interactions.

Cx43NT^∗37 channel gating

Lipophile-induced gating

A hallmark of GJChs, irrespective of Cx composition, is their increased tendency to close upon exposure to lipophilic agents that increase membrane fluidity. Burt and colleagues concluded that Cx43 GJChs were induced to close only by lipophiles that increase fluidity in the central region of the bilayer (particularly at the bilayer-channel interface (annular lipids)), thereby destabilizing the open state and stabilizing the closed state of the channel (37). Resistance of Cx43^∗NT37 channels to closure by halothane suggested that this region of the Cx43^∗NT37 channel may be more stable than in WT channels, a possibility supported by the high open probability observed for the chimera. Greater stability of the chimera’s open state could result in (at least) three ways. First, if the Cx37NT folds into the vestibule of the Cx43 PFD, it could support more stabilizing interactions in that position than the NT of Cx43 does. This possibility would not readily explain the larger unitary conductance despite increased cation selectivity of the chimera. Second, if the Cx37NT was unable to form a gating plug (possibly due to steric constraints), then, despite changes in global structure typical of membrane fluidizers, the channel might not close. This also seems unlikely because the channel can be closed by halothane; it just requires a higher concentration to effect closure. Third, the Cx37NT may not be able to fold back into the vestibule of the Cx43 PFD (lack of kinked structure), which would suggest that the normal role of the Cx43NT is to destabilize rather than stabilize the open state. Loss of a destabilizing influence and loss of the vestibule lining would then support a more stable open state and a larger conductance channel, which is what we observed for the Cx43^∗NT37 channel. To our knowledge, this chimera represents the first report of a connexin isoform that forms a stably open channel. However, it is interesting to note that Verma et al. (77) showed that ZP2519, a peptidomimetic, prevents low pH and octanol-induced uncoupling, the latter in a CT-dependent manner. These authors did no dose-response work with octanol to demonstrate that the channel could be closed when ZP2519 was present, but both the ZP2519 data and the Cx43^∗NT37 chimera data suggest open state stability is intimately dependent on PFD interactions with both the NT and CT.

V_j-induced gating

The conductance of GJs composed of Cx43 is reduced by an imposed V_j. Cx43 GJChs close (V₀ ∼60 mV) to a ∼35 pS subconductance state (residual state) in a CT-dependent manner (8). The P_o of Cx43 has been modeled to be ∼10% (78), similar to what we calculated for the cell pair shown in Fig. 3 A. Cx43^∗NT37 GJChs were less sensitive to V_j than Cx43WT GJChs, yet they continued to close to a residual state with large V_j. Moreover, their sensitivity to V_j was enhanced in the presence of halothane. These results are consistent with the view that the NT (and other parts of the PFD) comprises the voltage sensor and undergoes a conformational change that transduces a conformational change in the PFD (aided by halothane) to expose a binding site for the CT. Binding of the CT then stabilizes the conformation of the PFD transduced by the sensor, which we measure as a stable residual state. The V₀ of Cx37 channels is ∼30 mV (79), which raises the question of how the voltage sensor behaves in the Cx43^∗NT37 channel. Considering the NT portion of the voltage sensor, this domain may not be in the correct position to induce the conformational change of the PFD. Furthermore, without the appropriate NT, the PFD portion of the voltage sensor may be less sensitive to V_j. Alternatively, regardless of NT/PFD voltage sensitivity, transduction of a conformational change may be impaired by increased stability of the PFD in the open state. We cannot resolve with certainty these possibilities, but the increased sensitivity to V_j in the presence of halothane suggests resistance of the PFD to conformational changes in the absence of the lipophile.

Low pH-induced gating

As with V_j, the conductance of GJs composed of Cx43 is reduced with cytosolic acidification. Cx43 GJChs close completely (pK_a ∼6.6) at low pH in a CT-dependent manner. Without the CT, Cx43 channels are fairly resistant to closure by low pH, suggesting that while all cytosolic domains (NT, CL, CT) are exposed, low pH-induced changes in CT-PFD interaction are required to close the channel. That Cx43^∗NT37 channels do not close in response to low pH, despite retention of Cx43WT CT and PFD and exposure of these domains to low pH, suggests that although CT-PFD interaction still occurs (residual state), this interaction is unable to overcome the extremely stable open state of the channel conferred by the altered NT domain. We would predict that the chimera’s sensitivity to low pH would be enhanced by halothane, but this was not tested.

Gating synopsis

Our data indicate that compatible (Cx43) CT and CL domains are not sufficient to confer typical regulation by pH, V_j, and halothane on a Cx43 pore with the NT of Cx37. The two main possible explanations are that 1) CT-CL interaction also requires a compatible (Cx43) NT domain, or 2) the Cx37NT for Cx43NT exchange caused global structural changes that resulted in a high energy barrier between open and closed states, irrespective of gating trigger. Because gating by all types of triggers was compromised in the Cx43^∗NT37 chimera, we suggest that the global structure of the normal channel is determined by dynamic interactions of both the NT and CT with the channel’s PFD. These interactions enable gating, with modification of NT-PFD or CT-PFD interactions leading to increased stability of either the open or closed state.

Channel permeability, selectivity, and conductance

From both the crystal structure and MD simulations of the Cx26 channel (24, 27, 28), the NT domain is thought to line the vestibule of the channel where its bulk and electrical characteristics are well positioned to influence the size and charge selectivity of the open channel. Sequence comparison of the Cx43 and Cx37 NTs (Fig. 6) reveals eight mismatched residues in the 22 exchanged residues of the Cx43^∗NT37 chimera. In addition, the Cx37NT has a net negative charge compared to a neutral charge for the Cx43NT. The charged residues of the Cx37NT (E8, K9, D12, E16, (H17)) are thought to occupy one side of the Cx37NT α-helix, with hydrophobic residues lying on the opposite side (50). This difference in NT charge would certainly be expected to contribute to the cation preference of Cx37 channels. Interestingly, the roughly corresponding residues of Cx26 (Q7, S8, G11, K15, H16; net positive charge) were shown to face the cytoplasmic side of the pore funnel (24). In agreement with the previous data and other studies (49), the NT of Cx37 is sufficient to confer upon the Cx43 pore the low permeability, low permselectivity, and substantial cationic selectivity typical of Cx37.

That Cx43^∗NT37 channel conductance was nearer that of Cx43WT than Cx37 channels (47, 53, 80) is consistent with channel conductance being determined by the M1 domain (81, 82), in this case, that of Cx43. A small (residual) substate has been reported for channels of Cx26 (83), a protein with a very short (but nevertheless functional) CT domain. In contrast, the elimination of most of the CT domain from Cx40, Cx43, and Cx37 yields channels unable to reside in the residual configuration (7, 8, 16), which indicates that in the absence of a normal CT, the NT and CL alone do not readily produce this subconductive channel configuration. Furthermore, our CT chimera (Cx43^∗CT37) data suggest that compatible CT and CL domains are necessary to produce a residual state (17). Therefore, the presence of a V_j-induced substate and the transitions of intermediate amplitude at high V_j values seen in Cx43^∗NT37 GJChs are evidence of CT-dependent regulation of channel conductance.

Recapitulating, our data indicate that charge selectivity of GJChs is determined by the NT domain. However, unitary conductance of the fully open Cx43^∗NT37 channel, although somewhat bigger than that of parental Cx43, did not reach the values typical of the fully open Cx37 GJCh and therefore is likely determined by other regions of the channel. In addition, evidence of a V_j-induced residual state suggests regulation of conductance by the CT. Overall, ours and the referenced data suggest that the NT has a central role in establishing the essential molecular selectivity of the channel, whereas other (electrical and regulatory) properties are established by the concurrence of several elements, including domains of the connexin itself and its possible binding partners within the cell.

Conclusion

The behavior of Cx43^∗NT37 channels suggests a unified model of GJCh gating wherein dynamic interactions of the NT and CT with PFD elements alter protomer structure dynamically toward increased stability of either the open or closed state. The NTs likely form the channel’s six-membered gate, but their ability to form this gate depends on global changes of structure (e.g., radial expansion) permissive to their interaction; such changes of structure are induced by one or a combination of gating triggers. It appears that the CTs do not form the channel’s gate; instead, through interactions with other regions of the PFD, the CTs induce global changes of structure that are permissive to or stabilize gate formation by the NTs or result in narrowing or lengthening of the pore to affect permeability (subconductance states and/or size selectivity).

Author Contributions

All authors contributed to research design, analyzed and interpreted the data, and participated in writing of the article. T.K.P. performed molecular biology, cell and immunofluorescence work, and contributed to electrophysiology studies; J.F.E.V. performed electrophysiology and dye injection studies; J.M.B. supervised the project.

Editor: Joseph Mindell.

Supporting Citations

References (84, 85, 86, 87, 88, 89) appear in the Supporting Material.