Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential

Abstract

Nonjunctional membrane in many cells contains connexin gap junction hemichannels (or connexons) that can open to allow permeation of small molecules. Opening of Cx43 hemichannels is infrequent in normal extracellular Ca²⁺ and enhanced by low Ca²⁺, positive membrane potentials, and dephosphorylation of critical residues. Here we report that lowering intracellular redox potential increases Cx43 hemichannel open probability under otherwise normal conditions. We studied dye uptake and single-channel activity in HeLa cells transfected with wild-type Cx43, Cx43 with enhanced GFP attached to its C terminus (Cx43-EGFP), and Cx43 with enhanced GFP attached to its N terminus (EGFP-Cx43). Dithiothreitol [(DTT) 10 mM], a membrane permeant-reducing agent, increased the rate of dye uptake by cells expressing Cx43 and Cx43-EGFP, but not by parental cells or cells expressing EGFP-Cx43. Induced dye uptake was blocked by La³⁺, by a peptide gap junction and hemichannel blocker (gap 26), and by flufenamic acid. DTT increased Cx43-EGFP hemichannel opening at positive voltages. Bath application of reduced glutathione, a membrane impermeant-reducing agent, did not increase dye uptake, but glutathione in the recording pipette increased hemichannel opening at positive voltages, suggesting that it acted intracellularly. DTT caused little change in levels of surface Cx43 or Cx43-EGFP, or in intracellular pH. These findings suggest that lowering intracellular redox potential increases the opening of Cx43 and Cx43-EGFP hemichannels, possibly by action on cytoplasmic cysteine residues in the connexin C terminus.

Gap junction channels are formed by the union of two hemichannels or connexons, one from each of the apposed cells, and connecting the cytoplasm of adjacent cells, allowing a flux of small ions and molecules such as ATP, glucose, glutathione (GSH), cAMP, and IP₃ (1). Thus, gap junction channels allow electrical coupling, metabolic cooperation, and coordination of cell activities. Although some connexins have limited expression, Cx43 is expressed in many cell types, including astrocytes, fibroblasts, cardiomyocytes, and dendritic cells (2). Hemichannels are assembled in the endoplasmic reticulum and Golgi or post-Golgi compartments and are trafficked to the surface membrane. Hemichannels have been demonstrated in many cell types by immunocytochemistry, freeze-fracture EM, electrophysiological recording, dye uptake measurement, and/or biotinylation of cell surface proteins (3). Cx43 expressed in HeLa cells forms hemichannels with a small open probability (4). Hemichannel openings are increased by low extracellular Ca²⁺ (4, 5), which enhances release of small molecules such as ATP, glutamate, prostaglandin E2, and NAD⁺ (6), which may be paracrine signals. Opening of hemichannels may subserve a variety of functions in physiological and pathological conditions (3, 7), but their role is still controversial.

Metabolic stress induces activation of a large nonselective cationic channel in cardiomyocytes (8) and increases permeability to small molecules such as ethidium bromide (EtdBr) and Lucifer yellow in astrocytes (9, 10). These actions are ascribable to the opening of Cx43 hemichannels (8–10). In astrocytes, the increase in dye uptake can be inhibited with nordihydroguaiaretic acid, a blocker of lipoxygenases, trolox, melatonin (9, 11), reduced GSH ethyl ester, or dithiothreitol (DTT) (10). These agents are membrane permeant; they reduce the generation of free radicals, act as free radical scavengers, or reduce oxidized cysteines. The action of reducing agents suggests a role for redox potential in controlling hemichannel opening in cells under metabolic stress. Redox potential is known to modify the activity of different types of ion channels, including ryanodine receptors (12), P2X receptors (13), transient receptor potential (TRP) channels (14), potassium channels (15), and nonselective cation channels (16). At physiological cytosolic oxygen pressure, nitric oxide (NO) activates ryanodine receptors through S-nitrosylation of free cysteines of the channel subunit, but at atmospheric oxygen pressure, cysteine S-nitrosylation does not occur because cysteine residues are oxidized (12). In general, in vitro studies on functional gap junction channels and hemichannels have been performed at atmospheric oxygen pressure, an oxidizing condition that can increase the intracellular redox potential. Thus, regulation of connexin-based channels by change in redox potential of cells in vitro may have been overlooked.

In the present work, we used HeLa cells transfected with mouse Cx43, Cx43 with enhanced GFP attached to the C terminus (Cx43-EGFP), or Cx43 with enhanced GFP attached to the N terminus (EGFP-Cx43) to evaluate the effect of reducing agents on dye uptake and hemichannel opening. We found that DTT increases the rate of dye uptake at the resting redox potential in cells expressing Cx43 and Cx43-EGFP, but not in parental cells or cells expressing EGFP-Cx43. In whole-cell voltage clamp studies, DTT increased the open probability of Cx43-EGFP hemichannels at positive potentials. Additionally, GSH, a membrane impermeant, physiological-reducing agent, did not increase the rate of dye uptake when externally applied, but when included in the recording pipette increased openings at positive potentials, as did bath-applied DTT. DTT had little or no effect on the level or phosphorylation state of Cx43 in the surface membrane or on intracellular pH. These data indicate that the opening of Cx43 hemichannels is regulated by the intracellular redox potential, which may act through the cysteine residues in the C terminus of Cx43 or in an associated molecule.

Results

The Reducing Agent DTT Increases EtdBr Uptake in HeLa Cells Expressing Cx43-EGFP.

Because increased Cx43 hemichannel opening during metabolic inhibition is reversed by reducing agents (9, 10), we studied whether changes in redox potential affect hemichannel opening under normoxic conditions. We used HeLa cells transfected with Cx43-EGFP; these cells take up EtdBr uptake at the resting potential and exhibit unitary events at positive potentials, with conductance and pharmacological sensitivity predicted for Cx43 hemichannels (4). As reported for mixed cultures of parental and transfected cells (4), cells expressing more Cx43-EGFP showed faster EtdBr uptake (Fig. 1). Application of DTT (10 mM) rapidly increased the rate of dye uptake in cells showing Cx43-EGFP fluorescence (<3 min, records 1–3; Fig. 1B, bar at top; see also Fig. 2), but there was little increase in nonfluorescent cells (records 4–8). In Cx43-EGFP cells, the rate of EtdBr uptake after DTT application was increased from 0.29 ± 0.01 to 0.48 ± 0.02 a.u. per min (P < 0.001; ≈20 cells expressing high and moderate levels of Cx43-EGFP from 13 independent experiments). The rate of dye uptake was linearly related to the amount of Cx43-EGFP expressed (measured as fluorescence intensity) and was increased proportionally by DTT at each level of expression; the ratio of the slopes was ≈3 (Fig. 1C; correlation coefficients for control and after DTT were r² = 0.84 and 0.92, respectively; P < 0.001 for slopes before vs. after DTT). The increase in dye uptake was unexpected because DTT decreases dye uptake after dye uptake has been increased by metabolic inhibition (10).

Fig. 1.

DTT increases the rate of EtdBr uptake by Cx43-EGFP but not by parental cells; uptake is proportional to Cx43-EGFP expression. In mixed cultures of Cx43-EGFP and parental cells EtdBr (5 μM), uptake was measured every 100 sec as fluorescence emission of EtdBr binding to DNA (518 nm, AU of intensity). (A) Fluorescence micrograph of the measured cells showing EGFP expression. (B) Time course of EtdBr uptake in Cx43-EGFP cells (1–3 in A) and parental HeLa cells (4–8 in A) before and after application of 10 mM DTT (bar at top). (C) Rate of EtdBr uptake was proportional to Cx43-EGFP fluorescence for eight cells before (open circles) and after (filled circles) DTT treatment.

Fig. 2.

DTT-induced EtdBr uptake by Cx43-EGFP cells is prevented by hemichannel blockers, but not by blockers of P2X or TRPV1 channels. EGFP-Cx43 cells take up EtdBr at a rate like that of parental cells, but wild-type Cx43 does mediate uptake. EtdBr uptake was measured as for Fig. 1 in control conditions, after addition of 10 mM DTT (bar), and in A–D after later addition of a blocker (second bar). (A) In Cx43-EGFP cells, an increase in the rate of dye uptake caused by DTT was reversed by 200 μM La³⁺. (B) Similarly, 300-μM gap 26 reversed a DTT-induced increase in uptake. (C) Uptake induced by DTT was not affected by oATP, a P2X receptor blocker (300 μM was applied 30 min before uptake measurement and had no significant effect on basal uptake). (D) Uptake induced by DTT was not affected by capsazepine (CZP), a specific TRPV1 channel blocker (10 μM). (E) DTT had little effect on EtdBr uptake by EGFP-Cx43 cells (filled circles) or parental cells (open circles largely obscured by filled circles). (F) DTT increased uptake by cells expressing wild-type Cx43.

DTT at ≤1 mM had no effect on dye uptake [supporting information (SI) Fig. 7 A and D]; DTT at 10 and 30 mM increased the rate of dye uptake near maximally (by 67 ± 5% and 82 ± 6%; SI Fig. 7 B–D; n = 4, ≈20 cells per experiment; ∗∗∗, P < 0.001 vs. control; P > 0.5 for 10 vs. 30 mM).

DTT-Induced Increase in EtdBr Uptake Is Sensitive to Hemichannel Blockers.

If DTT increases uptake of EtdBr by opening Cx43-EGFP hemichannels, this effect should be inhibited by hemichannel blockers such as La³⁺ (4, 9, 17) and the gap 26 peptide, which has the same sequence as a region of the first extracellular loop of Cx43 (6). We measured basal uptake for 10 min and added DTT, which increased uptake. After ≈10 min, we added La⁺³ (200 μM; Fig. 2A, upper bar) or gap 26 (300 μM; Fig. 2B, upper bar), which rapidly reduced uptake to below that before DTT treatment, in La³⁺ to 65 ± 7% of the initial rate (P < 0.001; n = 9 experiments, 145 cells analyzed), and in gap 26 to 80 ± 5% of the initial rate (P < 0.01; n = 3, 60 cells analyzed). Thus, the DTT-induced increase in uptake and ≈20–30% of basal uptake were mediated by hemichannels. As shown in Fig. 1C, uptake depended on expression and varied among cells. Action of La³⁺ and gap 26 did not differ significantly (P > 0.05). Flufenamic acid (100 μM) also blocked dye uptake (data not shown).

To exclude uptake through a P2X receptor-dependent pathway or TRPV1 channels, we used specific blockers. oATP (300 μM), an irreversible P2X receptor blocker, applied 40 min before DTT (Fig. 2C; n = 3, 60 cells) or 10 min after DTT (data not shown) had no obvious effect on DTT-induced or basal uptake (data not shown). Capsazepine [(CZP) 10 μM], a TRPV1 channel blocker (18), did not affect DTT-induced dye uptake (Fig. 2D; n = 3, 60 cells).

To examine the possibility that Cx43-EGFP induces expression of other channels or transporters capable of mediating EtdBr uptake in HeLa cells, we used HeLa cells expressing EGFP-Cx43, which does not form functional hemichannels or channels, although it has an unmodified C terminus and does form plaques between cells (4). Basal uptake by EGFP-Cx43 cells (Fig. 2E, filled circles) did not differ from that of parental cells (open circles), and application of 10 mM DTT (bar) did not enhance EtdBr uptake by either type of cell (Fig. 2E; P > 0.05; four comparisons: transfected vs. parental cells, uptake after DTT vs. basal uptake; n = 3 experiments, 7 EGFP-Cx43 cells, 15 parental cells).

DTT Increases EtdBr Uptake by HeLa Cells Expressing Cx43.

Because the permeation through Cx43-EGFP hemichannels might be affected by the EGFP, we tested whether DTT affects EtdBr uptake in HeLa cells transfected with wild-type Cx43. Immunolabeling indicates that ≈100% of these cells express Cx43 (data not shown). Bath application of 10 mM DTT rapidly increased dye uptake (Fig. 2F; 0.22 ± 0.01 a.u. per min before DTT, 0.30 ± 0.01 a.u. per min after DTT; P < 0.001; n = 13, 251 cells analyzed). This increase is comparable to that in Cx43-EGFP cells (Fig. 1) and presumably depends on expression level.

DTT Increases Opening of Cx43-EGFP Hemichannels at Positive Voltages.

The opening of Cx43 hemichannels is increased at positive voltages (4), so we determined the effect of DTT on opening under these conditions. Polarizing a cell to +20 mV under whole-cell patch clamp did not induce opening of hemichannels with or without 10 mM DTT (Fig. 3A Left and Right). Voltage steps to +40 mV induced openings of a few hemichannels in cells under control conditions (1.5 ± 0.7 hemichannels per 40-sec pulse, the average of the maximum number of channels open simultaneously during the pulse; Fig. 3B Left) and after treatment with DTT (2.0 ± 0.9 hemichannels; P > 0.05; n = 5 cells; Fig. 3B Right). Polarizing to +60 mV further increased the number of openings, which were more frequent in cells treated with DTT (Fig. 3C Left and Right). The maximal number of hemichannels opening simultaneously was 3.0 ± 0.6 in control medium (3 in Fig. 3C Left) and 9.0 ± 0.8 after DTT (7 in Fig. 3C Right; n = 5 cells for each condition, four steps at each voltage, two before and two after DTT in each cell; P < 0.01). Hemichannel activity after DTT was blocked by 200 μM La³⁺ (Fig. 3D; n = 3) or 100 μM flufenamic acid (n = 3, data not shown). No channel activity was seen with parental cells at +60 mV after application of DTT (Fig. 3E; n = 5 cells).

Fig. 3.

DTT increases opening of Cx43-EGFP hemichannels at positive potentials. Unitary events were recorded by whole-cell voltage clamp. (A) At +20 mV, there were no unitary events in the absence (Left) or presence (Right) of 10 mM DTT in the bathing medium. (B) At +40 mV, there were a few openings before DTT (Left) and increased openings after DTT (Right). (C) At +60 mV, there were multiple openings before DTT (Left) and markedly increased openings after DTT (Right). (Left) Up to three hemichannels were open simultaneously. (Right) At least seven channels were open simultaneously later in the response. (D) La³⁺ (0.2 mM) blocked unitary activity at +60 mV in the presence of DTT. (E) No channel activity was seen in parental cells at +60 mV in the presence of DTT. (F) DTT decreased the latency of first opening at +40 mV and +60 mV (four pulses for each voltage before and after DTT in each of five cells (∗∗, P < 0.01 vs. control at each potential). (G) DTT increased the mean number of hemichannels open during the last 30 sec of pulses at +40 mV and +60 mV (∗, P < 0.05; ∗∗∗, P < 0.001).

DTT decreased the latency to the first opening at positive voltages. Before DTT, the latency was 11.3 ± 3.5 sec at +40 mV and 3.3 ± 1.6 sec at +60 mV (Fig. 3F; representative records in Fig. 3C Left and Right). After DTT application, the latency was 2.2 ± 0.4 sec at +40 mV and 1.5 ± 0.6 sec at +60 mV; both were significantly shorter than before DTT (Fig. 3F, ∗∗, P < 0.01; n = 5). DTT increased the mean number of open hemichannels during the last 30 sec of the response to positive voltage; at +40 mV, the increase was from 0.11 ± 0.01 to 0.26 ± 0.01 (Fig. 3G, ∗, P < 0.05; n = 5), and at +60 mV, the increase was from 0.30 ± 0.05 to 2.03 ± 0.22 (Fig. 3G, ∗∗∗, P < 0.001; n = 5).

The DTT-induced increase in the number of open channels is likely due to an increase in open probability at the given voltages because there was little change in surface expression (see below and Fig. 5). We do not know the extent to which the open probability of “active” hemichannels was increased or new hemichannels were recruited to the active population. The closure of channels on return to negative potentials appears too rapid to be due to internalization (data not shown).

Fig. 5.

Distribution and phosphorylation state of Cx43-EGFP and phosphorylation state of Cx43 are not affected by DTT. (A) Fluorescence micrographs of three Cx43-EGFP cells before and 60 min after 10 mM DTT (horizontal bar). Large intensely fluorescent gap junctions occupied intercellular appositions. Vesicles and nonjunctional surface also showed fluorescence. DTT had no apparent effect on Cx43-EGFP distribution. (B) Fluorescence intensity measured in the four boxes at cell margins in A showed no meaningful changes over time. (C) In Western blots from rat heart (lane 1), three phosphorylated bands (p2-p3 and a more slowly migrating band) and one more rapidly migrating, unphosphorylated band (np) were observed. In total homogenate from Cx43-EGFP HeLa cultures (50 μg of protein, lane 2), two bands of ≈65 and 70 kDa were detected, corresponding to Cx43-EGFP in different states of phosphorylation. Surface Cx43-EGFP isolated by biotinylation in control conditions (lane 3) and after 10-min treatment with 10 mM DTT (lane 4) showed the same two bands, although fainter, which differed little in density between lanes. Surface Cx43 from Cx43 HeLa cultures under control conditions (lane 5) and after 10-min treatment with 10 mM DTT (lane 6) showed similar bands in the two lanes (p2-p3, phosphorylated; np, nonphosphorylated). Molecular mass markers on the right (BSA, 80 kDa; ovalbumin, 49.1 kDa; carbonic anhydrase, 34.8 kDa).

DTT in the bath solution slightly but significantly changed the unitary conductance of hemichannels from 249 ± 3 ps (at +60 mV, n = 99 transitions) under control conditions (Fig. 3C Left) to 228 ± 3 ps (at +60 mV, n = 141 transitions) after DTT (n = 5 independent experiments; P < 0.001, nonparametric Student's t test). The effect of DTT in reducing unitary conductance is unexplained. The difference between ≈250 ps and the 220 ps value reported previously (4) is ascribable to minor differences in the media used (150 mM vs. 140 mM NaCl, 4 mM vs. 5.4 mM KCl, 1.2 mM vs. 1.8 mM CaCl₂).

Reducing Agents Act on Cx43-EGFP Hemichannels at an Intracellular Site.

To characterize the redox site(s) as intra- or extracellular, we bath applied GSH, a membrane impermeant-reducing agent. Extracellular application of 10 mM GSH at pH 7.4 (Fig. 4A, bar) had little effect on EtdBr uptake by Cx43-EGFP cells (P > 0.05; n = 6, 75 cells analyzed). We then performed whole-cell recording with a pipette solution containing either DTT or GSH. At +60 mV with 10 mM DTT in the pipette, there was a relatively high frequency of hemichannel openings similar to the effect of bath-applied DTT (Fig. 4B Left, n = 3; cf. Fig. 3C Right). At +60 mV, the effect of 10 mM GSH in the pipette was similar to that of DTT (Fig. 4B Right, n = 3). The reducing agents acted more rapidly than we could resolve. From the data in Fig. 2, the reducing agent might act in course of a single 60-sec positive step.

Fig. 4.

Intracellular DTT or GSH increases openings of Cx43-EGFP hemichannels at +60 mV. (A) In Cx43-EGFP cells, EtdBr uptake was not affected by bath application of 10 mM GSH. (B) (Left) In whole-cell patch clamp recording with 10 mM DTT in the pipette solution, four to five hemichannels opened in Cx43-EGFP cells at +60 mV. (Right) Ten millimolar GSH in the pipette solution had about the same effect as 10 mM DTT; four to five hemichannels opened in Cx43-EGFP cells held at +60 mV.

In conclusion, lowering the intracellular redox potential facilitates voltage-induced opening of Cx43-EGFP hemichannels. Lack of effect of bath-applied GSH on dye uptake indicates that the action of reducing agents on accessible, extracellular cysteines is not sufficient to increase hemichannel opening at the resting potential.

DTT Has Little Effect on Distribution of Cx43-EGFP or the Phosphorylation State of Cx43-EGFP or Cx43.

Fluorescence at the cell surface (rectangles in Fig. 5A) was monitored in four cells under control conditions and after DTT application. A total of nine cells were analyzed in three experiments. Cytoplasmic inclusions, presumably vesicles, exhibited some movement in control and in DTT, as did gap junction plaques (data not shown). Bath application of 10 mM DTT (bar) resulted in no consistent change in fluorescence of Cx43-EGFP at or near the surface (integrated fluorescence in the rectangles in Fig. 5A is plotted in Fig. 5B). This result suggests that DTT did not affect the amount of Cx43-EGFP in the surface membrane or acidify the cytoplasm because Cx43-EGFP fluorescence is pH-sensitive (19).

To confirm that DTT does not change the amount of Cx43 or Cx43-EGFP in the surface membrane, cell surface proteins were biotinylated, isolated with NeutrAvidin beads, and resolved by Western blotting with a C-terminal antibody (9). Western blots showed the typical forms of Cx43 present in rat heart homogenate (Fig. 5C, lane 1; the p2-p3 bands are phosphorylated and the NP band is not phosphorylated, or if it is phosphorylated, its mobility is not affected by dephosphorylation) (20). A homogenate of Cx43-EGFP cells showed an immunoreactive doublet of ≈60 and 70 kDa (Fig. 5C, lane 2); these bands probably correspond to two phosphorylated forms of Cx43-EGFP. Weaker bands were also present with electrophoretic mobility close to that of Cx43 in the rat heart. These bands might correspond to Cx43 released by proteolysis of Cx43-EGFP. The amount of Cx43-EGFP present on the cell surface was not significantly different under control conditions and after DTT treatment (Fig. 5C; 10 mM for 10 min; to 112 ± 2% of control, lanes 3 and 4; P > 0.05; n = 4). Similarly, DTT did not significantly increase the surface expression of Cx43 (Fig. 5C; to 115 ± 7% of control, lanes 5 and 6; P > 0.05; n = 3). Thus, the increase in the number of hemichannels in the surface membrane made little, if any, contribution to the increase in hemichannel opening induced by DTT.

Buffering Intracellular Ca²⁺ Does Not Alter DTT Action.

Because an increase in intracellular-free Ca²⁺ concentration may induce the opening of Cx32 hemichannels (21), we tested whether loading with the chelator BAPTA alters DTT's effects on EtdBr uptake by Cx43-EGFP cells. Addition of 10 mM DTT to cells loaded with BAPTA increased the rate of dye uptake from 0.32 ± 0.02 a.u. per min to 0.55 ± 0.04 a.u. per min (see SI Fig. 8 A and B; P < 0.001; n = 3, 60 cells analyzed), similar to the effect in cells without BAPTA.

DTT Action to Increase Dye Uptake in Normoxic Conditions Gradually Changes to a Decrease in Dye Uptake During Metabolic Inhibition.

The action of DTT on dye uptake is opposite in normoxic conditions and after prolonged metabolic inhibition (9, 10). We examined the transition from normoxic to the inhibited condition and applied DTT at various times after beginning metabolic inhibition [induced by iodoacetate (270 μM) and antimycin A (5 ng/ml)]. After 20 min of metabolic inhibition, DTT still increased the rate of dye uptake (to ≈260% of control; Fig. 6 A and D, ∗∗∗, P < 0.001; n = 4). After 30 min, DTT caused a much smaller increase (to ≈140% of control; Fig. 6 B and D, ∗, P < 0.05 vs. control; n = 4). After 40 min, DTT decreased the rate of dye uptake (to 40% of control; Fig. 6 C and D, ∗∗, P < 0.01; n = 4). The increase in dye uptake during metabolic inhibition can be accounted for by insertion of additional hemichannels into the surface membrane (10). The action of DTT to decrease dye uptake during metabolic inhibition is rapid enough to suggest an effect on open probability, rather than a reduction in surface expression. In contrast to the effect of DTT during metabolic inhibition, an increase in dye uptake by DTT in normoxic conditions is accompanied by little change in surface expression (Fig. 5C) and therefore is likely to be due to increased open probability.

Fig. 6.

The effect of DTT changes during metabolic inhibition. (A) Twenty minutes after addition of iodoacetate (270 μM) and antimycin A (5 ng/ml), DTT increased EtdBr uptake. (B) After ≈30 min of metabolic inhibition, DTT had less effect. (C) After ≈40 min, DTT decreased EtdBr uptake. (D) Quantitation of four experiments. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Discussion

Here we demonstrated that activity of Cx43-EGFP hemichannels in HeLa transfectants is sensitive to reduction in intracellular redox potential. We found that: (i) DTT increased dye uptake in Cx43 or Cx43-EGFP cells, but not in parental cells or cells expressing EGFP-Cx43, which does not form functional hemichannels; (ii) an increase in dye uptake was inhibited by hemichannel blockers; (iii) DTT increased hemichannel opening at positive voltages in Cx43-EGFP cells, but not in parental cells; (iv) GSH, a poorly membrane permeant, physiological-reducing molecule, enhanced hemichannel activity only when it was applied intracellularly; and (v) the DTT effect was not due to changes in distribution, abundance, or phosphorylation state (as assessed by electrophoretic mobility) of hemichannels or changes in intracellular pH (6). DTT presumably increased open probability of active channels and/or fraction of surface hemichannels that were active because it had at most a small effect on single-hemichannel conductance and surface expression.

Cx43 has nine cysteine residues, three in each of the two extracellular loops and three in the cytoplasmic C-terminal domain. Oxidation/reduction of intracellular cysteines may be involved in the regulation of the opening of Cx43-EGFP hemichannels. Hemichannels of other connexins with cysteine residues in the C terminus may also prove to be sensitive to redox potential; Cx30 and Cx56 have three and one C-terminal cysteines, respectively, whereas Cx26 and Cx45 have none. Not all cysteines may be sites for redox changes because of the influence of neighboring amino acids (22, 23). Cx32 has four cysteine residues in its C terminus, but Cx32 hemichannels expressed in HeLa cells are rather insensitive to DTT under normoxic conditions (H. Sánchez and J.C.S., unpublished data).

During metabolic inhibition, dye uptake ascribed to Cx43 hemichannels is increased, and most or all of the increase can be accounted for by increased surface expression. However, this increase is reversed by scavengers of reactive oxygen species, such as Trolox, melatonin (9, 11), DTT, and GSH ethyl ester (10). Extracellular GSH had no effect on hemichannel opening induced by metabolic inhibition (11), similar to its lack of action in normoxic conditions (Fig. 5), suggesting an intracellular site of action. Taken together, these data suggest that oxidation of Cx43 or a regulatory molecule during metabolic inhibition leads to increased surface expression and increased permeation through open hemichannels. Metabolic inhibition causes S-nitrosylation of Cx43, which may increase channel activity, and nitrosylation is reversed by DTT (10). The decrease in permeation by reducing agents during metabolic inhibition is in the opposite direction of the increase in permeation and opening induced by DTT under normoxic conditions, a difference not yet understood. The transition between increasing and decreasing permeation requires many minutes of metabolic inhibition (Fig. 6). Phosphorylation state may be relevant, and metabolic inhibition induces dephosphorylation of Cx43 hemichannels, as indicated by increased electrophoretic mobility.^¶ However, dephosphorylated Cx43 hemichannels remain open in a reconstituted system (25) and when expressed in Xenopus laevis oocytes (26). Trolox and DTT do not prevent dephosphorylation of Cx43 hemichannels during metabolic inhibition (10, 24). Moreover, cyclosporin A, a blocker of the phosphatase, calpain, reduces dephosphorylation during metabolic inhibition, but does not prevent dye uptake (11). These findings argue against a role of dephosphorylation in hemichannel opening during metabolic inhibition, although dephosphorylation of a critical subset of hemichannels cannot be excluded. Further study with mutation of specific cysteine residues and phosphorylation sites should resolve these issues.

How might reducing agents reverse the increase in permeability of hemichannels caused by oxidative stress during metabolic inhibition, yet increase opening under normoxic conditions? One possibility is that the same cysteine residues are substrates of different redox reactions, including formation and reduction of disulfide bonds, cysteine S-nitrosylation, and/or glutathionation (27). Alternatively, in different phosphorylation states, the same modifications could cause different conformational changes or the cysteines modified could be different.

Bath-applied DTT increased dye uptake proportionally to the expression of Cx43-EGFP (Fig. 1C). However, DTT did not lead to an obvious opening of hemichannels determined by whole-cell recording at potentials less than approximately +40 mV, although it increased EtdBr uptake in cells at their resting potential. The increase in Cx43-EGFP hemichannel activity at V_m ≥ 40 mV and the increase in dye uptake at the resting potential appear too rapid to be due to insertion of new hemichannels into the surface membrane. Moreover, the openings at positive potentials are rapidly reversed on returning the potential to inside negative values (data not shown). Finally, the increase in surface expression is minimal when evaluated by fluorescence and surface biotinylation (Fig. 5). At the resting potential, the hemichannel openings are very infrequent, but the upper limit of opening can still account for the dye uptake (4). However, change in cytoplasmic constituents associated with whole-cell recording might also result in reduced hemichannel opening at the resting potential. Recently, pannexin 1 (Px1), a member of the family of proteins forming gap junctions in invertebrates, has been implicated in the formation of active hemichannels in vertebrate cells (28, 29). Our hemichannels are unlikely to be pannexin-based because of the dependence on Cx43 expression, single-channel properties corresponding to those of Cx43 gap junctions, and sensitivity to octanol, La³⁺, and extracellular Ca²⁺ to which pannexin hemichannels are relatively insensitive (29). In addition and in agreement with a recent report (30), we did not detect pannexin 1 by Western blot analysis or immunofluorescence (data not shown).

In other cell types expressing Cx43, the normal intracellular redox potential may allow more hemichannel activity than that observed in HeLa cells and permit significant release of small molecules such as ATP, NAD⁺, glutamate, and PGE2 (6). Redox potential-sensing mechanisms are known to be relevant in physiological conditions, and cells can modify their intracellular redox potential in response to autocrine or paracrine signals. For example, EGF (31), PDGF (32), angiotensin II (33), and IL-1β (24) can induce production of reactive oxygen species. Because Cx43 is expressed by many cell types, modulation of its hemichannels by the intracellular redox potential may affect numerous cellular processes in which paracrine signaling occurs.

Materials and Methods

For additional methods, see SI Methods.

Cell Cultures.

Experiments were performed on HeLa cells (CCL-2; ATCC, Rockville, MD) transfected with cDNAs encoding mouse Cx43-EGFP or EGFP-Cx43 as described (4). Parental HeLa cells served as controls.

Time-Lapse Fluorescence Imaging.

Fluorescence images of cells in 150 mM NaCl/4 mM KCl/1.2 mM CaCl₂/5 mM Hepes at pH 7.4 plus 5 μM EtdBr were recorded every 30–90 sec by using an Olympus (Tokyo, Japan) BX 51W1I upright microscope with water-immersion lenses (528-nm excitation, 598-nm emission).

Electrophysiological Measurements.

Cells grown on coverslips (#0) were transferred to a chamber on an inverted Olympus IX-70 microscope. The pipette solution contained 130 mM KCl, 10 mM potassium aspartate, 0.26 mM CaCl₂, 2 mM EGTA, 5 mM tetraethylammonium-Cl, 1 mM MgCl₂, 3 mM MgATP, and 5 mM Hepes at pH 7.2. Single-channel conductance was calculated from single-channel currents, and the reversal potential, approximately −6 mV, was determined by extrapolation.

Immunoblots.

Proteins were analyzed by immunoblotting as described (10). Aliquots of cell lysates (50 μg of protein) or total biotinylated surface proteins were resuspended in a final concentration of 1× Laemmli sample buffer, separated on 8% SDS/PAGE, and electrotransferred to nitrocellulose sheets as described. Densitometric analyses of immunoblot signals were performed with National Institutes of Health IMAGE software.

Cell Surface Biotinylation.

Confluent cell cultures were treated with Sulfo-NHS-SS-biotin for 30 min at 4°C, followed by quenching with glycine. Cells were lysed, and NeutrAvidin beads were added followed by three rounds of centrifugation and washing. The resulting pellet was suspended in Laemmli buffer, which contained DTT that released labeled proteins from the beads, and centrifuged at 14,000 × g for 2 min. Cx43 in the supernatant was determined by immunoblotting.

Abbreviations

DTT

dithiothreitol

Cx43-EGFP

Cx43 with enhanced GFP attached to its C terminus

EGFP-Cx43

Cx43 with enhanced GFP attached to its N terminus

EtdBr

ethidium bromide

GSH

glutathione

TRP

transient receptor potential.