Although alkaline pH is known to trigger Ca2+ influx in diverse cells, no pH-sensitive Ca2+ channel has been identified. Here, we report that extracellular alkalinization induces opening of connexin 43 hemichannels (Cx43 HCs). Increasing extracellular pH from 7.4 to 8.5, in the presence of physiological Ca2+/Mg2+ concentrations, rapidly increased the ethidium uptake rate and open probability of HCs in Cx43 and Cx43EGFP HeLa transfectants (HeLa-Cx3 and HeLa-Cx43EGFP, respectively) but not in parental HeLa cells (HeLa-parental) lacking Cx43 HCs. The increase in ethidium uptake induced by pH 8.5 was not affected by raising the extracellular Ca2+ concentration from 1.8 to 10 mM but was inhibited by a connexin HC inhibitor (La3+). Probenecid, a pannexin HC blocker, had no effect. Extracellular alkalinization increased the intracellular Ca2+ levels only in cells expressing HCs. The above changes induced by extracellular alkalinization did not change the cellular distribution of Cx43, suggesting that HC activation occurs through a gating mechanism. Experiments on cells expressing a COOH-terminal truncated Cx43 mutant indicated that the effects of alkalinization on intracellular Ca2+ and ethidium uptake did not depend on the Cx43 C terminus. Moreover, purified dephosphorylated Cx43 HCs reconstituted in liposomes were Ca2+ permeable, suggesting that Ca2+ influx through Cx43 HCs could account for the elevation in intracellular Ca2+ elicited by extracellular alkalinization. These studies identify a membrane pathway for Ca2+ influx and provide a potential explanation for the activation of cellular events induced by extracellular alkalinization.
- alkaline pH
- calcium permeability
plasma membrane ca2+ channels are ubiquitous and serve an essential role in the regulation of varied cell processes such as muscle contraction, neurotransmitter release, epithelial secretion, maintenance of cell volume, and cell proliferation and migration (19, 36, 43). Three types of plasma membrane Ca2+ channels have been identified: voltage-gated, ligand-gated, and store-operated channels. However, the membrane pathway of the increased Ca2+ influx induced by alkaline extracellular pH has not been identified.
Connexin hemichannels (Cx HCs) are functional membrane hexameric channels present in diverse primary cell types such as astrocytes (11), myocardiocytes (30), kidney epithelial cells (70), osteocytes (10), hepatocytes (72), and neurons (63), as well as in cell lines derived from fibroblasts (24), endothelial cells (23), osteoblasts (57), and monocytes/macrophages (74), among others. Recent studies (22, 25, 26, 32, 41, 47) also support the presence of functional Cx HCs in tissues where they play relevant functions. Undocked HCs correspond structurally to half a gap junction channel, but instead of being at apposed cell membranes, as gap junction channels are (22), they are located at unapposed cell membrane domains and frequently show independent regulation and function from those of intercellular channels (11, 14, 22, 54, 60). Although HCs are believed to remain preferentially closed in cultured cells under resting conditions (4), their activation has been documented under experimental conditions of different physiological and pathophysiological natures (22, 60, 62). Hemichannels formed by connexin43 (Cx43), the most ubiquitous gap junction protein, participate in diverse cellular processes by mediating the transmembrane passage of small molecules such as ATP (32), NAD+ (7), glutamate (75), glutathione (51), PGE2 (10), and glucose (54).
A single channel conductance of ∼220 pS is characteristic of Cx43 HCs activated by positive membrane voltages (12) or by extracellular ligands such as proinflammatory cytokines (54) and FGF-1 (60), which increase their open probability. The predicted Cx43 HC pore diameter is rather large (68), and as expected, these HCs constitute nonselective pathways for ions and are permeable to molecules larger and more complex than small inorganic ions (22, 61, 37, 38). In theory, Ca2+ permeation through Cx43 HCs could explain their involvement in several physiological and pathological responses associated with HC activation (11, 14, 15, 24, 25, 26, 30, 38, 44, 47, 50, 58, 60). However, extracellular divalent cations such as Ca2+ and Mg2+ reduce the open probability of Cx HCs (18, 37, 67), and the open probability of all Cx HCs so far studied is rapidly enhanced by removal of extracellular divalent cations (22, 37, 60). Although the blocking effect of divalent cations may prevent or limit Ca2+ fluxes through HCs, the divalent cation effect seems to be overcome under certain circumstances. For example, under resting conditions, physiological extracellular Ca2+ concentration ([Ca2+]) reduce the open probability of Cx43 HCs (12), limiting Ca2+ inflow. However, Cx43 HC activation occurs in the presence of extracellular divalent cations under conditions that mimic ischemic, proinflammatory, or proliferative conditions (11, 14, 15, 24, 26, 30, 34, 47, 54, 60, 65) or treatment with nitric oxide donors (52) or reducing agents (53). Exposure of rabbit cardiomyocytes to metabolic inhibitors for 60 min increases the intracellular [Na+] and [Ca2+], effects reduced ∼50% by the nonselective HC and gap junction blockers halothane and heptanol (38). Furthermore, the increase in intracellular [Ca2+] in isolated rat neonatal cardiomyocytes exposed to oxygen and glucose deprivation is inhibited by Gap26, a Cx mimetic peptide blocker of Cx43 HCs (65). The above results suggest the occurrence of Ca2+ influx via HCs. However, after metabolic stress, intracellular Na+ accumulation reduces the Ca2+ efflux through Na+/Ca2+ exchange and also via plasma membrane Ca2+-ATPase (PMCA), promoting intracellular Ca2+ accumulation (9). In addition, dantrolene, a blocker of Ca2+ release from endoplasmic reticulum also inhibits the elevation in intracellular [Ca2+] induced by ischemic-like conditions (65). Therefore, a role of Ca2+ influx through activated HCs is possible but remains uncertain.
In Cx43 HeLa transfectants treated with FGF-1 or 4Br-A23187, a Ca2+ ionophore, cell permeabilization is directly related to an increase in surface Cx43 levels (60). The FGF-1 effect requires and favors an increase in intracellular [Ca2+], suggesting that Cx43 HCs are permeable to this ion (60). Although Ca2+ influx associated with HC expression is required for the metabolic inhibition-induced increase in surface HC levels, as well as for the resulting cell permeabilization to ethidium in HeLa cells transfected with Cx32 (58), there is no demonstration of Cx43 HC permeability to Ca2+ in cells exposed to alkaline pH.
Here, using HeLa transfectants, electrophysiological measurements, and confocal time-lapse fluorescence recordings, we show that HCs formed by Cx43 are activated by extracellular alkalinization. Our data strongly suggest that activated Cx43 HCs are permeable to Ca2+ and that the Cx43 COOH-terminal domain is not essential for sensing rises in extracellular pH.
MATERIALS AND METHODS
Reagents and solutions.
We used a previously characterized polyclonal anti-Cx43 antibody (6, 60) and a monoclonal antibody against an epitope of the NH2-terminal tail of mouse Cx43 (anti-NT1 antibody) generously provided by Dr. Paul Lampe (Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA; Ref. 66). An influenza hemagglutinin tag (cat. no. 71–5500) antibody was obtained from Zymed Laboratories (Carlsbad, CA). Ethidium bromide (Etd), LaCl3 (La3+), probenecid, and calf intestine alkaline phosphatase immobilized on agarose were obtained from Sigma-Aldrich (St. Louis, MO). Fluo 4-AM, fura red-AM, and calcium green-2 were from Invitrogen (Carlsbad, CA). The l-α-phosphatidylcholine and l-α-phosphatidylserine were purchased from Avanti Polar Lipids (Alabaster, Al), and the detergent n-decyl-β-d-maltopyranoside was from Antrace (Maumee, OH). The composition of the recording solution was as follows (in mM): NaCl (150), KCl (5.4), CaCl2 (1.8), MgCl2 (1), glucose (5), and HEPES (5), pH 7.4 or 8.5. The Etd used for dye uptake experiments was prepared in water and diluted to a 5 μM final concentration in recording solution. A 100 mM (100×) probenecid, stock solution was prepared in distilled water. LaCl3 was dissolved in recording solution at 100× final concentration. All stock solutions were diluted to final concentrations in recording solution prepared in sterile and filtered water (W3500; Sigma-Aldrich).
HeLa cell cultures.
Previously described parental HeLa cells (CCL-2; ATCC, Rockville, MD) and HeLa cells stably transfected with mouse Cx43 (HeLa-Cx43) were kindly provided by Dr. Klaus Willecke (Bonn University, Bonn, Germany; Ref. 20). A variant Cx43 truncated at amino acid 257 (Cx43Δ257) was generated by Dr. Agustín D. Martínez (Universidad de Valparaiso, Valparaiso, Chile) and was previously described by Schalper et al. (60). The vector used for Cx43 transfections was pcDNA1/Neo (Invitrogen), which contains the cytomegalovirus promoter, polyadenylation signal, and a gene conferring resistance to G418. The vector alone was transfected into HeLa cells to study the effect of transfection itself on the membrane permeability increase elicited by extracellular alkalinization. HeLa cells were transfected with 1 μg pcDNA I/Neo using the calcium phosphate transfection protocol. Twenty-four hours after removal of the DNA precipitate, G418 (300 μg/ml) was added to the culture medium. The transformed cells were resistant to G418 but did not express detectable levels of Cx43 (mock-transfected HeLa cells). HeLa cells expressing Cx43 with EGFP fused to its C terminus (HeLa-Cx43EGFP) were kindly provided by Dr. Feliksas Bukauskas (Albert Einstein College of Medicine, New York, NY) and have been described previously (8). Cells expressing Cx43Δ257 were selected with 300 μg/ml G418 and identified using an antibody directed against a 9 amino-acid influenza hemagglutinin tag inserted in the construct (60). All cells were cultured in DMEM supplemented with 10% FBS and kept at 37°C in a 5% CO2/95% atmosphere at nearly 100% relative humidity. Cells expressing Cx43EGFP were selected with 300 μg/ml G418 and identified by their fluorescence emission at 530 nm, as described (60). Cells were seeded onto plastic Falcon Petri dishes (Becton Dickinson, Los Angeles, CA) or onto sterile glass coverslips placed on the bottom of plastic culture dishes. Cx expression was tested by indirect immunofluorescence (Fig. 1) or fluorescence intensity of the EGFP-tagged protein (Fig. 4). Cell cultures showing membrane staining in >95% of the cells were used in all experiments.
Extracellular alkalinization protocol.
Extracellular alkalinization was achieved by superfusing cells with recording solution (see above) adjusted to pH 8.5 with NaOH/HCl. The pH of all solutions was titrated to the desired pH, at room temperature, and all solutions were prepared 30 min before each experiment. Subconfluent (<50%) and 48-h serum-starved HeLa cells were used since previous studies (60) revealed the highest HC-mediated response to FGF-1 under these conditions. Since relatively small extracellular osmolarity increases (e.g., 25 mosmol/l) activate Cx43 HCs in cardiomyocytes and Cx43-transfected HeLa cells (31), the osmolarity of control (pH 7.4) and alkaline solutions (pHs 7.8 and 8.5) was measured using a vapor pressure osmometer (Wescor 5520; Vapro, Logan, UT). As expected due to the low buffer concentration (5 mM HEPES), there was no significant difference between the osmolarity of the solutions (319 ± 5 mosmol/l at pH 7.4 vs. 323 ± 7 mosmol/l at pH 8.5; P > 0.05).
Time-lapse fluorescence imaging and intracellular [Ca2+].
For time-lapse experiments, cells plated on glass coverslips were washed twice with recording solution and placed into a custom-made perfusion chamber on the stage of a Zeiss LSM 510 confocal microscope (Thornwood, NY), using a ×40 1.2 NA objective lens, essentially as described previously (56). Excitation was accomplished through the 488-nm line of a krypton/argon laser. In all cases, measurements and data analyses were performed using the same microscope and camera settings, at which neither autofluorescence nor background signals were detectable. Excitation intensity was low to prevent photobleaching, which was not observed when the cells were illuminated for a few minutes. For the Etd uptake experiments, cells were superfused with recording solution containing 1 μM Etd and fluorescence emission between 565 and 615 nm was recorded every 4 s in regions of interest of different cells. Results from 4–20 cells per experiment were averaged. For the assessment of cytosolic Ca2+ changes, cells were loaded for 45 min with the acetoxymethyl ester forms of fluo 4 (6 μM) or fura red (6 μM), at 37°C, followed by a 30-min deesterification period. After three washes in recording solution, cells were left to stabilize at 37°C for 5 min before recordings started. Emission was recorded between 500 and 530 nm for fluo 4, or above 650 nm for fura red, with acquisition rates of 1–5 Hz. Changes in fluorescence are presented as percentage of the difference between peak fluorescence and baseline fluorescence. In another set of time-lapse Etd uptake experiments, cells plated onto glass coverslips were washed twice with recording solution and incubated in 5 μM Etd, and fluorescence intensity was recorded in regions of interest of different cells with a water immersion Olympus 51W1I upright microscope. Images were captured with a Q Imaging model Retiga 13001 fast cooled monochromatic digital camera (12-bit; Qimaging, Burnaby, BC, Canada) every 20 s (exposure time = 30 ms; gain = 0.5) and Metafluor software (version 6.2R5; Universal Imaging, Downingtown, PA) was used for image analysis and fluorescence quantification. Slopes were calculated using Microsoft Excel software and expressed as arbitrary units per minute.
Cells seeded on glass coverslips were placed onto a custom made chamber mounted on the stage of an inverted Olympus IX-51 microscope. The extracellular bath solution contained (in mM) 140 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 2 BaCl2, and 10 HEPES, pH 7.4. For whole-cell patch-clamp experiments, the pipette solution contained (in mM) 130 CsCl, 10 AspNa, 0.26 CaCl2, 1 MgCl2, 2 EGTA, 7 TEA-Cl, and 5 HEPES, pH 7.2. Whole cell currents were recorded as described previously (60). Patch electrodes were made from borosilicate glass capillaries using a Flaming/Brown micropipette puller (P-87, Sutter Instruments, Union City, CA). The tip resistance was 5–10 MΩ when filled with pipette solution. Currents were filtered at 1 kHz and sampled at 5 kHz. Then, records were filtered with a digital low pass filter of 0.5 kHz. Data acquisition and analysis were performed using pClamp 9 (Axon Instruments, Novato, CA).
Surface protein biotinylation and Western blot analysis.
Cell cultures seeded on 100-mm culture dishes were washed three times with recording solution. Then, 3 ml of sulfo-NHS-SS-biotin (0.5 mg/ml) were added to each dish and incubation proceeded for 30 min at 4°C. Cells were then washed three times with recording solution containing 15 mM glycine, pH 8.0, to quench unreacted biotin. Afterward, cells were harvested by scraping with a rubber policeman in the presence of protease and phosphatase inhibitors (as for Western blot analysis, see below) and centrifuged at 4 g for 2 min. Pellets were sonicated with a Microson Sonicator ultrasonic homogenizer (Misonix, Farmingdale, NY) on ice in 50 μl lysis buffer containing protease (200 μg/ml soybean trypsin inhibitor, 1 mg/ml benzamidine, 1 mg/ml aminocaproic acid, and 2 mM PMSF) and phosphatase (20 mM Na4P2O7 and 100 mM NaF) inhibitors. The immobilized NeutrAvidin (1 μl of NeutrAvidin per 3 μg of biotinylated protein) was added to each sample, and the mixture was maintained for 1 h at 4°C. Then, 1 ml of binding buffer (recording solution at, pH 7.2, plus 0.1% SDS and 1% NP-40) was added, mixed by soft vortexing, and centrifuged at 4 g for 2 min. Then, the supernatant was removed and 40 μl recording solution at pH 2.8 plus 0.1 M glycine were added to the pellet, mixed gently, and centrifuged again at 4 g for 2 min. The supernatant was removed, and pH was adjusted to 7.4 immediately by adding 10 μl of 1 M Tris, pH 7.4. Protein levels of cell lysates were measured with the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA). Samples were then analyzed by immunoblotting. Briefly, aliquots of cell lysates (100 μg of protein) were resuspended in 1× Laemmli sample buffer, separated on 8% SDS-PAGE, and electrotransferred to nitrocellulose sheets. Nonspecific protein binding was blocked by incubating the nitrocellulose sheets in 5% nonfat milk prepared in PBS for 60 min, and then blots were incubated with primary polyclonal anti-Cx43, anti-hemagglutinin, or anti-Cx43 NH2-terminal antibody overnight at 4°C, followed by six 20-min PBS washes. Each primary antibody was diluted in 5% nonfat milk in PBS. Blots were incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5,000 in 5% nonfat milk in PBS). Antigen-antibody complexes were detected by ECL using the SuperSignal kit according to the manufacturer's instructions. Resulting immunoblot signals were scanned and densitometric analyses were performed using the Scion Image software (Scion, Frederick, MD). Relative levels of surface Cxs were determined by densitometry.
Cells plated onto glass coverslips were fixed with 70% ethanol for 20 min at −20°C and incubated in blocking solution (70% goat serum, 30% PBS, and 1% BSA) for 30 min at room temperature. Then, cells were incubated overnight at 4°C with a rabbit anti-Cx43 polyclonal primary antibody (1:500 in blocking solution) characterized and described previously (6). Preparations were then washed six times with PBS for 5 min, incubated with the GARF-conjugated anti-rabbit IgG antibody for 1 h at 4°C, and submitted again to six 5-min PBS washes at room temperature. Finally, coverslips were mounted using Fluoromont G and examined with an Olympus FW100 confocal microscope equipped with a ×100 1.4 NA lens. Preparations were excited at 488 nm, and emission between 505 and 525 nm was collected.
Ca2+ measurements in liposome preparations containing purified Cx43-HCs.
Determinations of Ca2+ transport were performed on liposomes containing purified Cx43 HCs prepared as described previously (3). Briefly, Cx43 with a COOH-terminal His tag, expressed using a baculovirus/insect-cell expression system, was purified based on its affinity for Ni2+. The purified protein, solubilized in n-decyl-β-d-maltopyranoside, was dephosphorylated with alkaline phosphatase immobilized on agarose to assure that HCs were permeable and then reconstituted in l-α-phosphatidylcholine:l-α-phosphatidylserine, 2:1 molar ratio. Unilamellar liposomes and proteoliposomes of <100 nm in diameter were generated by dialysis/extrusion (3), and their size was confirmed by dynamic light scattering measurements (BI 200SM; Brookhaven Instruments, Holtsville, NY). Calcium green-2 was trapped inside the liposomes and proteoliposomes during the extrusion process at a concentration of 40 mM, in 10 mM KCl, 5 mM EDTA, and 10 mM HEPES, pH 7.4. After a 10-fold dilution in the same buffer, but without calcium green-2, the extraliposomal probe was removed by gel filtration. Fluorescence from calcium green-2, which increases upon Ca2+ binding (17), was measured on a Hitachi F-7000 spectrofluorimeter (Tokyo, Japan) at excitation and emission wavelengths of 503 and 536 nm, respectively.
Data are presented as means ± SE, and n represents the number of independent experiments. Means were compared using paired and unpaired t-tests for two group variables and one-way ANOVA followed by a Bonferroni posttest in multiple data sets. Differences between proportions were assessed using descriptive statistics. Differences were considered significant at P < 0.05. Statistics were performed using Microsoft Excel and the GraphPad Prism 4.0 (2003).
It has previously been shown that HeLa cells transfected with Cx43, but not parental HeLa cells, express functional Cx HCs (12, 14, 60, 71). In agreement with those studies, we detected similar Cx43 reactivity in the cell membrane and intracellular compartments of isolated HeLa-Cx43 cells, using confocal immunofluorescence, while no membrane signal was detected in parental cells (Fig. 1A). The presence of Cx43 forming undocked HCs at the HeLa-Cx43 cell membrane was confirmed by surface protein biotinylation and Western blot analyses (see Fig. 6). No evident differences in either cell morphology or Cx43 immunoreactivity pattern were detected in HeLa-Cx43 cells exposed for up to 10 min to pH 8.5, compared with control cells exposed to extracellular pH 7.4 (Fig. 1A).
In HeLa-Cx43 and -parental cells under control conditions (pH 7.4 and 1.8 mM Ca2+/1 mM Mg2+), the Etd uptake rate was <0.04 arbitrary units/min (Fig. 1, B and C). Exposure to extracellular pH 8.5 rapidly increased (<15 s) the rate of Etd uptake (Fig. 1B) by <75% in Cx43-transfected HeLa cells (Fig. 1C; P < 0.01). When HeLa-Cx43 were reexposed to control extracellular pH, the Etd uptake rate returned to baseline levels, whereas reexposure to pH 8.5 increased the Etd uptake rate, similar to the first alkaline exposure, indicating complete reversibility of the response (Fig. 1B). In contrast, no change in the Etd uptake rate was observed in HeLa-parental cells subjected to the same experimental protocol (Fig. 1, B and C; P > 0.05), or in HeLa cells transfected with the vector used for Cx43 expression, without insert (not shown), indicating that the above responses were not induced by the transfection protocol. Cells exposed to pH 7.8 showed an Etd uptake rate with a clear tendency above the control cells at pH 7.4 or parental cells exposed to pH 8.5 (Fig. 1C). Notably, the increase in the Etd uptake rate induced by pH 8.5 in HeLa-Cx43 cells was abolished by 200 μM La3+, a Cx HC blocker (48), but remained unaffected by the application of 1 mM probenecid, a Px1 HC inhibitor (59) (Fig. 1D).
To further characterize the pathway through which exposure to extracellular pH 8.5 increases the membrane permeability of HeLa-Cx43 cells, we used the whole cell voltage-clamp technique to study macroscopic and microscopic currents. As previously shown (60), at V = 0 mV the current value was zero (Fig. 2A), characteristic of a nonselective membrane channel such as Cx and Px HCs (2, 12). Exposure to pH 8.5 increased the total current ∼1.5 times (Fig. 2, A and B). Moreover, rectangular voltage pulses from 0 mV to +80 mV (5-s duration) evoked current transitions that were more numerous and presented a much shorter “on time” (latency; 0.8 ± 0.4 s and 2.2 ± 0.5 s, respectively) at pH 8.5 than at pH 7.4 (Fig. 2C). When the membrane potential was brought to −20 mV, a tail current was readily apparent, with unitary events with longer “off time” in cells exposed to pH 8.5, compared with control cells (7.5 ± 1.2 s and 0.9 ± 0.6 s, respectively; Fig. 2D, compare pH 7.4 and 8.5). Both, the current evoked by voltage pulses from 0 to +80 mV and the tail unitary currents generated by voltage pulse from +80 to −20 mV were abolished by 200 μM La3+ (Fig. 2, C and D, bottom traces, respectively), a blocker of Cx HCs that does not affect Px HCs (48). The tail current generated with a step from +80 mV to −20 mV presented unitary events with similar single-channel conductances of ∼220 pS at pHs 7.4 and 8.5 (Fig. 2D, insets), indicative of Cx43 HCs (12), and different from Px1 HCs (475–550 pS) (2). The combined data strongly suggest that Cx43 HCs are the pathway for the increased Etd uptake upon alkalinization.
Since extracellular alkalinization induces cell death associated with increased intracellular [Ca2+] (39), variations in intracellular Ca2+ levels upon Cx43 HC activation by extracellular pH 8.5 were monitored in cells preloaded with the Ca2+ indicator fluo 4. Extracellular alkalinization increased the peak fluo 4 fluorescence in HeLa-Cx43 cells but did not affect it in parental cells (Fig. 3A; P < 0.01). The increase in Ca2+-dependent fluorescence observed in HeLa-Cx43 cells was rapid (<15 s), reaching a steady-state after <2 min (Fig. 3B). As expected for influx through activated Cx43 HCs, raising the transmembrane Ca2+ gradient by increasing extracellular [Ca2+] to 10 mM promoted an additional increase in the alkalinization-triggered fluo 4 fluorescence in HeLa-Cx43 cells, reaching a new steady-state value after ∼45 s (Fig. 3, A and B; P < 0.01). The rate of increase in fluo 4 emission upon extracellular alkalinization was ∼2.3-fold higher in 10 vs. 1.8 mM extracellular [Ca2+] (Fig. 3, B and C; P < 0.001). Reexposure of HeLa-Cx43 cells to the control solution (pH 7.4, 1.8 mM Ca2+/1 mM Mg2+) lowered the fluo 4 signal to levels that approached control values (Fig. 3B). In contrast to the results in HeLa-Cx43, exposure of HeLa-parental cells to pH 8.5, either in the presence of 1.8 or 10 mM extracellular [Ca2+], had little effect on both fluo 4 fluorescence emission and its rate of increase (Fig. 3, A and C; P > 0.05). Interestingly, despite the reduction in the open probability induced by extracellular Ca2+ at physiological extracellular pH (12), the membrane permeability to Etd in cells exposed to pH 8.5 in the presence of 10 mM Ca2+ was still higher than that in control cells and was completely inhibited by 200 μM La3+ (Supplemental Fig. 1; Supplemental Material for this article is available online at the Am J Physiol-Cell Physiol website). In agreement with the low open probability of Cx43 HCs in cells exposed to physiological [Ca2+] at pH 7.4, increasing extracellular [Ca2+] to 10 mM did not reduce Etd uptake (compare Fig. 1C and Supplemental Fig. 1). Notably, the increase in Etd uptake recorded at pH 8.5 in the presence of 1.8 mM and 10 mM extracellular Ca2+ was similar, indicating that the increase in Etd uptake induced by extracellular alkalinization is insensitive to Ca2+.
To further evaluate the Cx43 dependence in the responses described above, intracellular Ca2+ levels were measured using fura red in cocultures of HeLa-parental and HeLa-Cx43EGFP cells; the latter have been shown to produce functional HCs (12, 60). Different to most Ca2+ indicators, fluorescence of fura red decreases upon Ca2+ binding. As shown in Fig. 4A, extracellular alkalinization in the presence of 1.8 mM extracellular [Ca2+] induced a larger increase in intracellular Ca2+ levels in Cx43EGFP-expressing cells [Cx43EGFP (+)] than in HeLa-parental cells [Cx43EGFP (−)]. Moreover, the alkaline-induced response was much higher after the extracellular [Ca2+] was raised to 10 mM only in Cx43EGFP-expressing cells (Fig. 4A; P < 0.01). In the latter, exposure to 10 mM extracellular Ca2+ at pH 8.5 rapidly decreased the fura red signal (<15 s), reaching a lower steady-state value <60% below the baseline (Fig. 4B, black circles). At extracellular pH 8.5, reduction of extracellular [Ca2+] to 1.8 mM decreased the fura red signal, reaching a new steady state <30% below baseline, revealing again the extracellular Ca2+ dependence of the observed fura red fluorescence responses. Only a small reduction in fura red fluorescence (<10%) occurred in parental cells during the recorded time period (Fig. 4, A and B, white circles). The decrease in fura red fluorescence rate elicited by extracellular alkalinization in the presence of 1.8 mM Ca2+ was inversely related to the Cx43EGFP fluorescence intensity (Fig. 4C; r2 = 0.84; P < 0.001; n = 10). A similar relationship was found in cells exposed to pH 8.5 in the presence of 10 mM extracellular [Ca2+] (r2 = 0.94; P < 0.001; n = 10; data not shown).
Since the increase in membrane permeability to Etd could result from a rapid rise in the number of HCs in the cell membrane, we tested if exposure to extracellular alkalinization affects the amount of Cx43EGFP at the cell membrane. To this end, we measured the fluorescence intensity of different membrane domains using confocal microscopy and found that exposure to pH 8.5 did not affect the fluorescence over a time course comparable to that of the Etd uptake and intracellular Ca2+ measurements described above (Fig. 5). Moreover, we found that 12-min exposure to a saline solution containing 10 mM Ca2+ did not affect the Cx43EGFP fluorescence present in the cell membrane (Fig. 5C).
The COOH-terminal domain of Cx43 has been involved in the sensitivity to intracellular pH and “chemical” gating of Cx43-based gap junction channels and HCs (16). To test its potential role in the effect of extracellular alkalinization, we studied HeLa cells expressing a truncated Cx43 (Cx43Δ257), lacking most of the C-terminal domain. These cells have been shown to form HCs that are activated by exposure to Ca2+/Mg2+-free solutions (60). To further confirm the previous findings, surface membrane proteins of HeLa-Cx43Δ257 and HeLa-Cx43 cells were biotinylated and levels of Cxs were evaluated by Western blot analysis of pulled down proteins, using an antibody directed against an NH2-terminal epitope. Blots revealed the presence of both proteins at the cell surface, at comparable levels (Fig. 6A; P > 0.05). However, in HeLa-Cx43Δ257 cells, the rate of fluo 4 signal increase was ∼1.8-fold that of HeLa-Cx43 cells (Fig. 6B; P < 0.05). We have previously shown that the HC-mediated membrane permeability to Etd of Cx43 and Cx43Δ257 transfectants under resting conditions is similar (60). In summary, as opposed to the gating of Cx43 intercellular channels by intracellular pH (16), the effect of extracellular alkalinization on Cx43 HCs is not abolished by the COOH-terminal domain truncation.
As shown above, sustained activation of Cx43 HCs by exposure to pH 8.5 elicited a higher steady-state intracellular Ca2+ level (Fig. 3B) but induced a continuous increase in Etd uptake rate (Fig. 1B). The differences in kinetics could be explained by a change in the HC selectivity or by the presence of mechanism(s) with capacity to control the raise in intracellular [Ca2+]. In support of the latter possibility, we found that treatment with 1 mM (Fig. 7A) or 5 mM (not shown) sodium orthovanadate, which blocks PMCA pumps (13), results in a higher plateau in the fluo 4 fluorescence induced by pH 8.5 in cells expressing Cx43 HCs (Fig. 7A; P < 0.05). Exposure to pH 8.5 in the presence of sodium orthovanadate also caused a larger reduction in fura red fluorescence of HeLa-Cx43EGFP cells [Fig. 7B, Cx43EGFP (+)], not observed in HeLa-parental cells [Fig. 7B, Cx43EGFP (−)]. These results suggest that the level of the increase in [Ca2+] induced by extracellular alkalinization is limited by mechanisms that include the PMCA pumps.
Previous studies (29) have shown that Cx43 affects the expression of several proteins, including plasma membrane channels and transporters. Therefore, we decided to confirm the Ca2+ permeability of Cx43 HCs more directly. To this end, we performed experiments with purified Cx43 HCs, dephosphorylated and reconstituted in liposomes. Liposomes with or without Cx43 HCs, with the Ca2+-sensitive probe calcium green-2 trapped inside, were prepared as described previously (3). A ∼1.8-fold larger increase in the calcium green-2 fluorescence emission occurred upon increasing extraliposomal [Ca2+] from 5 to 20 μM in the presence of dephosphorylated Cx43 HCs (Fig. 8; P < 0.02). The increases in fluorescence emission in liposomes lacking Cx43 HCs are due to extraliposomal calcium green-2 not removed by gel filtration (see materials and methods). These results show that HCs formed by dephosphorylated Cx43 are permeable to Ca2+.
In the present work, we show that Cx43 HCs are rapidly and reversibly activated by extracellular alkalinization to pH 8.5, despite the presence of high extracellular [Ca2+], which blocks HCs. Activation of Cx43 HCs was directly related to increased Ca2+ influx, leading to a higher steady-state intracellular Ca2+ level, limited in magnitude by the activity of PMCA pumps, as expected from the increase in intracellular Ca2+ signal. Purified Cx43 HCs, dephosphorylated and reconstituted in liposomes, allow passage of Ca2+, providing direct evidence for Ca2+ permeation through activated HCs. In addition, we show that the COOH-terminal domain of Cx43 is not involved in sensing or mediating the HC response to extracellular alkalinization and that it might even have a negative regulatory effect. This contrasts with the inhibitory effect of intracellular acidification, which depends on the presence of the COOH-terminal domain. Our data strongly suggest that Cx43 HCs activated by extracellular alkalinization are permeable to Ca2+ and that they are likely to play a relevant role controlling intracellular Ca2+ homeostasis under physiological and pathological conditions in which extracellular alkalinization occurs.
Cx43 HC activation by extracellular pH 8.5.
In addition to Cxs, pannexins (Pxs) can form functional HCs in vertebrate cells (64). Cx and Px HCs are permeable to ions and small molecules and are often endogenously coexpressed in diverse cell types (64). The use of heterologous expression systems and pharmacological approaches has been valuable to discriminate between the relative contributions of Cx and Px HCs (48, 59, 61). On the one hand, although HeLa cells have detectable Px1 levels (60), neither surface Px1 expression nor Px1 HC activity has been detected (28, 60), i.e., functional Px1 HCs are not expressed in these cells. Moreover, we found that extracellular pH 8.5 does not induce Px1 HC activation at the cell membrane since probenecid, a Px1 HC blocker (48), did not prevent the alkalinization-induced HC activation. On the other hand, HeLa cells transfected with Cx43, but not parental HeLa cells, express functional Cx HCs (14, 60, 71). In support of the latter, we found that increased Etd uptake induced by pH 8.5 was abolished by La3+, a known inhibitor of Cx HCs (59). These observations support the notion that HeLa cells do not express functional HCs formed by Pxs or endogenous Cxs. In agreement with the above observations, electrophysiological measurements revealed the presence of nonselective membrane channels (zero current at V = 0 mV) and only discrete single channels events of ∼220 pS in cells expressing Cx43EGFP HCs as described previously (12).
Exposure to extracellular solution with pH 8.5 rapidly increased the Cx HC-dependent Etd uptake rate, revealing activation of Cx43 HCs. This was not due to an increase in HC levels at the cell membrane because we did not detect changes cellular distribution of wild-type Cx43 (using immunofluorescence and confocal detection) or changes in surface level of Cx43EGFP (using confocal detection) in response to pH 8.5. Therefore, it is likely that alkaline pH activates a gating mechanism leading to opening of Cx43 HC. In agreement with this interpretation, activation of current events at pH 8.5 occurred with much less delay and the “off time” of single channels was much longer than at pH 7.4, which are indications of an increase in open probability. Therefore, the increase in HC activity was due to an increase in open probability and not to increased levels of HCs at the cell membrane, as observed in cells under metabolic inhibition or FGF-1 treatment (52, 58, 60). Similar to our observations on Cx43 HCs, Cx26 and Cx46 HCs expressed in Xenopus oocytes are rapidly activated by extracellular alkalinization (55, 69), and for Cx26 HCs, atomic force microscopy data suggest an increase in the external HC-pore diameter (76).
Intracellular acidification reduces the activity of intercellular gap-junction channels formed by diverse Cxs (27, 49), and intracellular alkalinization increases the open probability of gap junction channels formed by Cx45 (46) or Cx57 (45). One putative domain involved in sensing the intracellular pH changes is the cytoplasmic COOH-terminal domain, which, at least in Cx43-based intercellular channels, has been proposed to act as part of a gating “ball-and-chain” mechanism (16). Our findings show that the COOH-terminal domain of Cx43 is not required for the alkalinization-induced HC activation. We found that at comparable plasma membrane protein levels, the increase in Etd uptake was more pronounced in cells expressing Cx43Δ257 compared with those expressing wild-type Cx43. These data suggest that truncated HCs are released from the inhibition mediated by the COOH-terminal domain, expected from a “ball-and-chain” mechanism activated by acid pH, which possibly interferes with or reduces HC activation induced by pH 8.5. Our results clearly show that the Cx43 HC response to extracellular alkalinization cannot be explained by a “ball-and-chain” mechanism that involves the COOH-terminal domain. The findings suggest involvement of other protein domain(s). Further mutagenesis studies will be needed to identify the domains involved in the gating of Cx43 HCs by extracellular alkalinization.
Cx43 HCs permeability to Ca2+.
The close relationship between Cx43EGFP levels and fura red fluorescence intensity changes upon exposure to pH 8.5 indicates that cells expressing more HCs also display more Ca2+ influx. In contrast, no significant increase in intracellular Ca2+ levels was elicited by extracellular alkalinization in parental cells lacking functional HCs (HeLa-parental cells or mock-transfected HeLa cells). These findings, together with the higher Cx43 HC activity revealed by Etd uptake measurements, suggest that Cx43 HCs allow Ca2+ influx.
The rise in intracellular [Ca2+] upon activation of HCs by extracellular alkalinization is compatible with the notion of passive Ca2+ influx through HCs; the elevation in intracellular Ca2+ signal was enhanced by increases in the Ca2+ electrochemical driving force. In support of Ca2+ influx through HCs, an increase in intracellular [Ca2+] was reported in thapsigargin-treated HeLa cells expressing Cx43 upon switching from a Ca2+-free to a saline solution containing 1.3 mM [Ca2+] (71). In that study, the rise in intracellular [Ca2+] was inhibited by Gap26, a Cx mimetic peptide known to inhibit Cx43 HCs, suggesting Cx43 HCs as a pathway for Ca2+ influx (71). In the present work, we demonstrated that purified and dephosphorylated Cx43, known to form functional HCs in liposomes (3), increased intraliposomal Ca2+ upon increasing extra-liposomal [Ca2+], indicating that Cx43 HCs provide a transmembrane Ca2+ pathway. Quantitative correlation between the data in cells and proteoliposomes is difficult because the HC responses are likely affected by posttranslational modifications and/or the modulatory effect of intracellular binding partners found in vivo. Nevertheless, all of our data support the notion of Ca2+-permeable Cx43 HCs.
Increase in Cx43 HC activity has been demonstrated previously in the presence of physiological extracellular concentration of divalent cations. Examples include astrocytes (11), cardiomyocytes (30) and kidney epithelial cells (70) under metabolic inhibition, cortical astrocytes subjected to hypoxia-reoxygenation (44), and cells treated with proliferative or proinflammatory agents (14, 54, 60). Here, we found that activation of Cx43 HCs induced by extracellular alkalinization occurs even in the presence of 10 mM extracellular [Ca2+], showing that Cx43 HC gating by alkaline pH overcomes the reduction in open probability induced by extracellular divalent cations. Alternatively, a conformational change induced by pH 8.5 could affect the open probability through reduction in the HC extracellular Ca2+ sensitivity.
Since Cx43 is ubiquitously expressed (62), it is possible that Cx43 HCs serve as a membrane pathway for Ca2+ influx in a variety of cell types, mediating diverse Ca2+-dependent physiological and pathological responses. Accordingly, it was recently reported that FGF-1 increases the intracellular [Ca2+] in Cx43-transfected HeLa cells but not in parental cells (60). The tissues in which opening of Cx43 HCs by extracellular alkalinization could play relevant roles are the biliary and intestinal epithelium, where Cx43 is expressed (5, 25) and cells are exposed to pH values close to or above 8.0 (21, 35). Ca2+ influx via Cx43 HCs might also explain the alkalinization-induced increase in intracellular [Ca2+] through Ca2+ influx in vascular endothelial and vascular smooth muscle cells (1, 42, 73). Moreover, Ca2+ influx through Cx43 HCs could participate in a range of undesired effects of the hypocalcemia that results from alkalemia. This hypocalcemia has been historically attributed to an increase in binding of Ca2+ to proteins such as albumin (40). However, increased Ca2+ influx through Cx43 HCs may also play a role. Finally, persistent Ca2+ influx via Cx43 HCs might be related to alkalosis-induced cell death, a well-established phenomenon (39). Further work will be needed to define the range of circumstances in which this putative Ca2+ influx pathway contributes to intracellular signaling and homeostasis.
This work was partially supported by Fondo Nacional de Desarrollo Científico y Tecnológico Grants 1070591 (to J. C. Sáez), Núcleo Milenio P04/030-F (to J. C. Sáez), Anillo ACT 71 (to J. C. Sáez), FONDEF D07I1086 (to J. C. Sáez and K. A. Schalper), Texas Advanced Research Program 010674-0046-2007 (to G. A. Altenberg), NIH R01 GM-79629 (to G. A. Altenberg), and NIH R01GM-079629-03S1 (to G. A. Altenberg). K. A. Schalper received a Comisión Nacional de Investigación Científica y Tecnológica and Pontificia Universidad Católica fellowship for graduate studies.
No conflicts of interest, financial or otherwise, are declared by the author(s).
The data of this work were presented by Dr. Kurt Schalper as partial fulfillment of the requirements to obtain the degree of Ph.D. in Medical Sciences at the Pontificia Universidad Católica de Chile.
- Copyright © 2010 the American Physiological Society