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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Cell Biology and Neuroscience, University of California, Riverside, California
Submitted 22 December 2006 ; accepted in final form 24 September 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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257), cell-to-cell coupling was similarly decreased by an elevation of [Ca2+]i (IC50 of 310 nM Ca2+) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca2+]i regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43. gap junction; calmodulin; HeLa cells; lens epithelium
Given the ubiquity of gap junctions in all mammalian tissues except for mature skeletal muscle, there have been extensive studies examining the regulation of gap junction permeability, which has been shown to be regulated by protein kinase-catalyzed phosphorylation, Ca2+, intracellular pH, and transjunctional voltage (6, 28). Protein kinase-mediated phosphorylation of connexins is now recognized to provide a key mechanism for regulating both the gating and turnover of gap junction channels (32). Many of the connexins have been shown to be phosphorylated in their COOH-terminal regions, which contain multiple protein kinase consensus sequences. Cx43, the most widely distributed connexin, has been shown to be phosphorylated both in vitro and in intact cells by a number of known and unknown protein kinases. Previous studies from our laboratory have demonstrated that receptor activation results in the inhibition of Cx43 gap junctions via a PKC-dependent signal-transduction pathway in both Cx43-transfected HeLa cells (36) and a lens epithelial primary cell culture system (10, 37) in which serine 368 in the COOH terminus of Cx43 appears to play an essential role.
It has been over 30 years since it was first reported that increased cytosolic Ca2+ concentration ([Ca2+]i) can close gap junctions (46, 54) and 25 years since the first studies demonstrated [Ca2+]i regulation of lens gap junctions (30), which was subsequently shown to be mediated by the Ca2+ receptor calmodulin (CaM) (51). Although there are many intracellular receptors for Ca2+, the Ca2+-binding protein CaM appears to be the major receptor for this ion in most tissues, and this protein has also been identified in the lens (34, 35). Inhibition of lens fiber cell-to-cell communication by elevated [Ca2+]i was first demonstrated as an increased internal electrical resistance (21) that was prevented by preincubation with CaM antagonists. Elevated [Ca2+]i also inhibits cell-to-cell communication in both bovine (12) and sheep (10) lens primary cell cultures.
Critical control of [Ca2+]i is essential for maintaining both electrical coupling (55) and transparency (16) of the lens. Furthermore, like other tissues, lens cells maintain a submicromolar [Ca2+]i (8, 16, 30), the importance of which is illustrated by the observation that loss of lens Ca2+ homeostasis is a key factor in the early steps that result in cataract formation (16). Thus, in the most common form of human cataract, in which opacity occurs in the lens cortex, total lens Ca2+ is significantly elevated, as it is in most animal cataract models (16, 26). It has been proposed that cells in the nuclear regions of the lens are able to exchange Ca2+ with the fiber cells via their extensive gap junction connections to the cortical layer of cells (16, 21), although this may be an oversimplification considering the restricted range of action of [Ca2+]i (2, 39).
Our group has demonstrated that cell-to-cell communication was half-maximally inhibited at 360 nM [Ca2+]i in primary cultures of lens cultures (10) and was prevented by preincubation of lens cultures with CaM antagonists (37). The rapid onset of this inhibition (within seconds) suggests that this is mediated by a direct interaction of CaM with one or more of the lens connexins rather than being mediated via the action of a CaM-dependent protein kinase. Indeed, Peracchia et al. (51) have demonstrated that CaM directly gates Cx32-containing gap junctions, and Törok et al. (58) have identified two distinct CaM binding amino acid sequences in Cx32. The goal of the study reported here was to determine whether the Ca2+/CaM-dependent inhibition of lens cell-to-cell communication was mediated in part by Cx43. This was accomplished with Cx43-transfected HeLa cells because these cells have been shown to provide an excellent system to examine the properties of homotypic gap junctions (17, 18). Our results support the thesis that the Ca2+-dependent inhibition of Cx43 gap junctions results from the interaction of CaM with a cytoplasmic portion of Cx43 that excludes the bulk of its COOH-terminal region.
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MATERIALS AND METHODS |
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257 were a gift from Dr. Erika TenBroek (University of Minnesota, Minneapolis, MN; currently at Medtronics, Minneapolis, MN). Characterized FBS was purchased from Hyclone (Logan, UT). G418 (geneticin) and hygromycin B were obtained from Invitrogen (Carlsbad, CA). Fura 2-AM and Alexa fluor 594 (AF594) were from Molecular Probes (Eugene, OR). DMEM, HBSS without or with divalent cations, and all other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.
Cell culture.
HeLa cells were grown to confluence on sterilized glass coverslips in 35-mm culture dishes in DMEM (with 44 mM NaHCO3, pH 7.2) supplemented with 10% vol/vol FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Stable transfectants were grown under antibiotic selection as follows: for Cx43, 0.4 mg/ml G418; for Cx43257, 0.2 mg/ml hygromycin B. All cells were grown in a humidified 37°C incubator with 5% CO2.
Ca2+ determination and image analysis. Confluent monolayers of cells grown on glass coverslips were loaded with the Ca2+ indicator fura 2-AM (1 µM) in 2 ml HBSS buffer with divalent cations (containing 1.8 mM Ca2+, with added 10 mM HEPES, 5 mM NaHCO3, pH 7.2) and then transferred to a microincubation chamber (model MSC-TD, Harvard Apparatus, Holliston, MA) as described previously (37). Imaging of intracellular Ca2+ was performed with a Nikon TE300 (Nikon, Melville, NY) inverted microscope equipped with Nikon filter blocks for fura 2 emission and AF594 optics (Chroma Technology, Rockingham, VT), a Metaltek filter wheel (Metaltek Instruments, Raleigh, NC) housing excitation filters for fura 2, a 75-W xenon short arc lamp, and a Hamamatsu charge-coupled device digital camera (Hamamatsu, Bridgewater, NJ) and supported on a vibration isolation table (Technical Manufacturing, Peabody, MA). [Ca2+]i was measured ratiometrically (ratio of 340-nm to 380-nm wavelength) with fura 2 throughout each experiment in the injected cell and the cells adjacent to the injected cell, and Ca2+ concentrations were determined as described previously (10). Data collection was accomplished with MetaFluor software (version 3.5, Universal Imaging, Downington, PA).
Microinjection and assessment of gap junctional communication.
Micropipettes (borosilicate glass capillaries: 1 mm OD, 0.75 mm ID, 100-µm internal microfilament; Dagan, Minneapolis, MN) were pulled on a Flaming/Brown-type pipette puller (P-87; Sutter Instruments, Novato, CA). Micropipettes had tip diameters of <1 µm and resistances of 
100–300 M
when filled with AF594 dissolved in deionized water. The micropipette was positioned with a low-drift hydraulic micromanipulator (MW-3; Narishige, Greenvale, NY), and AF594 was microinjected with a micropipette lowered 1–3 µm beyond apparent contact with the cell's plasma membrane, which impaled the cell. Pipettes contained a 100 µM solution of AF594 dissolved in distilled deionized water. AF594 was injected with a train of 5-ms current pulses applied every 100 ms for 60 s (3-s total injection time) at ambient temperature. If the micropipette became plugged, it was replaced with a new micropipette, and the data from such a partial injection were excluded from the analysis. Current was generated with a Duo 773 (World Precision Instruments, Sarasota, FL). Current duration, magnitude, and polarity were controlled with an A310 Accupulser pulse generator (World Precision Instruments). Digitized images of AF594 cell-to-cell transfer were recorded 5 min after the iontophoretic injection of fluorescent dye.
Manipulation of [Ca2+]i with ionomycin.
We generated a sustained elevation in [Ca2+]i in Cx43-transfected HeLa cells by adding 1 µM ionomycin to the medium and then 2 min later increasing the extracellular Ca2+ concentration ([Ca2+]o) from 1.8 to 21.8 mM. Cx43257-transfected HeLa cells were more sensitive than wild-type Cx43-transfected HeLa cells to the membrane-soluble polyether antibiotic ionomycin in that in some experiments a significant elevation of [Ca2+]i could be accomplished by the addition of 1 µM ionomycin in normal (1.8 mM) [Ca2+]o medium. A subsequent increase in [Ca2+]o to 21.8 mM elevated [Ca2+]i in all Cx43257-transfected HeLa cells, albeit [Ca2+]i greater than 0.5 µM typically resulted in a loss of plasma membrane plasticity of Cx43257-transfected HeLa cells, such that it was now not possible to achieve a successful microinjection of AF594 dye in these cells.
[Ca2+]i of the cells was monitored after ionomycin addition to ensure [Ca2+]i stabilized such that only monolayers of cells whose [Ca2+]i had stabilized within 2 min of inducing elevated [Ca2+]i were used in these experiments. Cell-to-cell and culture-to culture variabilities in the resultant [Ca2+]i measured after the addition of ionomycin was likely due to differences in the effective concentration of this ionophore that incorporated into the cell plasma membranes.
Inhibition of CaM was accomplished by preincubating both wild-type Cx43 and Cx43257-transfected HeLa cells with calmidazolium (10 or 2 µM, respectively) for 20 min before the addition of ionomycin; this concentration has been shown previously to effectively inhibit gap junctions (48).
Depletion of intracellular Ca2+ stores and stimulation of capacitative Ca2+ entry in Cx43-transfected HeLa cells. Capacitative Ca2+ entry in Cx43-transfected HeLa cells was induced by incubating fura 2-loaded cells in nominally Ca2+-free buffer (HBSS without divalent cations; 10 nM Ca2+) for 5 min, followed by emptying intracellular Ca2+ stores by the addition of 250 nM thapsigargin (Tg; 10 min) and then raising [Ca2+]o to 10 mM. Confirmation that the intracellular Ca2+ stores were in fact emptied was determined before dye injection by the inability to elicit a Ca2+ wave following mechanical stimulation of these cells (44).
Data analysis. Data are presented as either representative single experiments or means ± SE of the mean based on pooled data from several experiments. All data are presented as the raw number of cells, showing communication for clarity and ease of comparison among figures and with data from other published reports. Where appropriate, differences among treatments were determined by ANOVA with means separated by the Student's t-test; differences from control value were considered significant at P < 0.01.
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RESULTS |
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257) (see Fig. 1) express functional gap junctions capable of cell-to-cell transfer of the dye AF594 that can be inhibited by the gap junction inhibitors carbenoxolone and 1-octanol (36).
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360 nM) in different cell types (11) and that this inhibition is not PKC dependent (Fig. 3A) or CaM kinase II mediated (Fig. 3B) (see also Ref. 37). However, as demonstrated previously with lens cell cultures, preincubation of Cx43-transfected HeLa cells with the CaM inhibitor calmidazolium (10 µM) prevented this [Ca2+]i-dependent inhibition of Cx43-mediated cell-to-cell dye transfer {20.8 ± 0.7 cells (n = 5) in the presence of calmidazolium and elevated [Ca2+]i (810 ± 110 nM Ca2+) vs. 21.2 ± 0.8 cells (n = 9) in the presence of resting [Ca2+]i (150 ± 100 nM Ca2+)} (Fig. 2D). Thus this Ca2+-dependent inhibition of gap junctions in Cx43-transfected HeLa cells would appear to be CaM mediated.
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257-transfected HeLa cells reduces cell-to-cell dye transfer.
Although it has long been recognized that Ca2+ affects inhibition of Cx43-containing gap junctions (47), to date there is no information as to which amino acid region(s) of Cx43 is responsible for mediating this action of CaM. It has been proposed that one CaM-interacting region resides in the COOH terminus of Cx43 (1, 50–52, 58). Therefore, the Cx43 COOH-terminal truncation mutant, Cx43257, stably expressed in HeLa cells, was utilized to test the hypothesis that the COOH-terminal region from amino acid residues 257–382 of Cx43 contains not only the pH-sensing domain (15) and multiple phosphorylation sites (31) but also the region mediating the action of CaM on Cx43. When [Ca2+]i was significantly elevated (from 40 ± 10 to 560 ± 120 nM) in these cells expressing the truncated form of Cx43, there was a significant decrease in cell-to-cell dye transfer (from 19.3 ± 0.4 cells, n = 15, to 3.4 ± 0.8 cells, n = 4) (Fig. 5B); the IC50 for this inhibition was 310 nM Ca2+ (Fig. 5A). One difference between the wild-type Cx43 and this truncated mutant is that, in contrast to the wild-type Cx43-transfected HeLa cells, ionomycin addition alone was frequently sufficient to elevate [Ca2+]i in the Cx43257 cells in 1.8 mM [Ca2+]o (i.e., it did not always require the further addition of 20 mM [Ca2+]o); all data with Cx43257 cells demonstrating significantly elevated [Ca2+]i was pooled (see Fig. 5B). This minor difference in method likely reflects a modest difference in the regulation of these two forms of Cx43 (41a). However, irrespective of the [Ca2+]o, cell-to-cell dye transfer was only inhibited when [Ca2+]i was greater than 350 nM before dye injection (Fig. 5, A and B).
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257-transfected HeLa cells with the CaM inhibitor calmidazolium prevents Ca2+-dependent inhibition of cell-to-cell dye transfer.
To determine whether the Ca2+-dependent decrease in cell-to-cell dye transfer between Cx43257-transfected HeLa cells was also CaM mediated, cells were preincubated with calmidazolium (2 µM) before elevation of [Ca2+]i with ionomycin (1 µM). In these experiments, CaM inhibitor and ionophore concentrations were reduced compared with those used with wild-type Cx43-transfected HeLa cells, as the Cx43257 transfectant was more sensitive to these agents than the wild-type Cx43. Higher concentrations of these agents resulted in cell lysis and the release of fura 2, which we have shown previously (10) to be an effective reporter of the viability of HeLa cells. As with wild-type Cx43, addition of the CaM inhibitor calmidazolium alone did not significantly change cell-to-cell dye transfer (18.7 ± 0.3 cells, n = 9) or elevate [Ca2+]i beyond normal resting levels for HeLa cells (80 ± 20 nM [Ca2+]i, n = 9) (Fig. 5C). However, when [Ca2+]i was significantly elevated after the addition of 1 µM ionomycin (350 ± 70 nM [Ca2+]i, n = 5) in the presence of the CaM inhibitor calmidazolium, the Ca2+-dependent inhibition of cell-to-cell dye transfer was prevented (19.8 ± 0.8 cells, n = 5, vs. 19.3 ± 0.4 cells in resting [Ca2+]i in the absence of calmidazolium) (Fig. 5, A and C). |
DISCUSSION |
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Although it has long been known that Ca2+ can close gap junctions (46, 54), the Ca2+ sensitivity of this uncoupling has varied widely from the nanomolar range (12, 33, 43, 49) to a low micromolar (41, 54) to hundreds of micromolar range (3, 19, 56). Furthermore, although [Ca2+]i can increase to the hundreds of nanomolar after application of agonists that elevate [Ca2+]i, and this is frequently associated with cell-to-cell Ca2+ waves in gap junction-coupled cells, as shown by others (44), these transient elevations in [Ca2+]i do not result in an inhibition of gap junctions between either lens epithelial cells (9) or Cx43-transfected HeLa cells (36).
Recently, Dakin et al. (13) examined the effect of three protocols that raised [Ca2+]i on gap junction communication of a fluorescent dye between Cx43-expressing human primary fibroblasts isolated from the foreskin of newborns. These protocols included activation of cell surface receptors with agonists such as bradykinin or histamine, elevation of [Ca2+]i with a Ca2+ ionophore, and stimulation of capacitative Ca2+ influx by emptying intracellular stores. Although these three protocols all raised [Ca2+]i to micromolar concentrations, these increases with agonist or ionophore were transient and were without effect on gap junctional coupling. That the addition of ionophore did not result in an inhibition of gap junctional communication in these cells (13) is likely because [Ca2+]o was not elevated to the concentrations that we report here as being required for the inhibition of Cx43 gap junctions (Fig. 2). Dakin et al. (13) demonstrated that stimulation of capacitative Ca2+ influx strongly inhibited gap junction coupling between these fibroblasts, which contrasts with results reported here (Fig. 4B) in which stimulation of capacitative Ca2+ influx by pretreatment of cells with Tg and then elevation of [Ca2+]o did not affect cell-to-cell communication between Cx43-transfected HeLa cells. Possible explanations for the differences between these two reports may include the different assay protocols for measuring cell-to-cell dye transfer, differences in the dye molecules used, or the different cell types. Furthermore, in both studies, Ca2+ reporter molecules reported averaged cytosolic [Ca2+]i results and therefore may not have reflected the true Ca2+ concentration adjacent to the gap junctions at the plasma membrane (14, 40). The explanation for these differences is not obvious and may only be resolved by the use of different protocols to elevate [Ca2+]i, coupled with different experimental approaches to measure cell-to-cell communication with a single defined cell type that contains a single connexin type.
The data reported here are very relevant in terms of the recent data of Gao et al. (22) who demonstrated that, whereas [Ca2+]i was as high as 700 nM in the central regions of the lens, it decreased to 300 nM at the lens surface. Thus, in the region where Cx43 is found in the outer equatorial epithelial cells, [Ca2+]i would be below that required to completely inhibit gap junctions that are composed primarily of Cx43. Thus, as proposed by Gao et al., Ca2+ would move from the interior regions of the lens via gap junctions to be transported out of the lens by the Ca2+-ATPase pump and Na+/Ca2+ exchanger present in the equatorial epithelial cells (45).
The data reported here, in which the Cx43 truncation mutant Cx43257 exhibited a similar Ca2+ sensitivity and CaM dependency as wild-type Cx43 (compare Fig. 2 with Fig. 5), indicate that this regulation is likely mediated via the interaction of CaM with a cytoplasmic region of Cx43 that excludes the bulk of its COOH-terminal region. Although this region has not been previously implicated in mediating the actions of CaM on gap junctions, Törok et al. (58) have examined the interaction of Cx32-derived peptides with CaM and have identified a high-affinity CaM-binding region in the NH2 terminus of this protein that is absent in Cx43. In addition, in support of the data reported here, these authors identified a region in the COOH-terminal sequence of Cx43 (residues 314–325) that did not associate with CaM; these authors did not examine other regions of Cx43 as possible candidate CaM receptors.
The precise mechanism by which CaM inhibits Cx43-mediated cell-to-cell communication remains to be defined, but there is significant evidence that CaM interacts directly with a number of connexins (7, 58, 63). There are two models by which CaM could therefore inhibit gap junctions. In the first model, cytosolic apo-CaM would bind Ca2+ after sustained elevation of [Ca2+]i and the Ca2+/CaM complex would associate with a cytoplasmic portion of Cx43, inducing a conformational change in Cx43 such that cell-to-cell coupling mediated by Cx43 gap junctions is inhibited (Fig. 6A). This is the more general mechanism by which CaM has been shown to regulate the function of many CaM-dependent processes (27, 59). Alternatively, like a number of membrane channel proteins (20), apo-CaM would be constitutively associated with Cx43 in a Ca2+-independent manner. When [Ca2+]i is elevated after a sustained elevation of [Ca2+]i, the Cx43-bound CaM induces a conformational change in Cx43 such that cell-to-cell coupling mediated by Cx43 gap junctions is now inhibited (Fig. 6B).
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257 exhibits the same Ca2+ sensitivity and CaM dependency as wild-type Cx43, indicate that this regulation is likely mediated via the interaction of CaM with a cytoplasmic portion of Cx43 that excludes the bulk of its COOH-terminal region. Identifying whether CaM mediates the inhibition of Cx43 via its direct interaction with Cx43 in either a Ca2+-dependent or Ca2+-independent manner or whether it acts via a member of a regulatory pathway that in turn regulates Cx43 permeability is the goal of ongoing investigations in this laboratory. |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) or presence (
) of the CaM inhibitor CDZ; Ca2+ IC50 = 0.36 µM in the absence of CDZ. C: summary data of control experiments. Hatched bars, [Ca2+]i; solid bars, no. of coupled cells. Av, average. For left bars (no additions), [Ca2+]i = 0.11 ± 0.01 µM and dye transferred to 20.1 ± 0.5 cells (n = 14). With ionomycin, [Ca2+]i = 0.19 ± 0.03 µM and dye transferred to 20.9 ± 0.7 cells (n = 8). With 21.8 mM [Ca2+]o, [Ca2+]i = 0.05 ± 0.01 µM and dye transferred to 21.0 ± 0.6 cells (n = 3). With elevated [Ca2+]i (ionomycin + 21.8 mM [Ca2+]o), [Ca2+]i = 0.62 ± 0.1 µM and dye transferred to 4.0 ± 0.4 cells (n = 5). D: summary data for CaM inhibition experiments. Hatched bars, [Ca2+]i; solid bars, no. of coupled cells. For CDZ only, [Ca2+]i = 0.15 ± 0.1 µM and dye transferred to 21.2 ± 0.8 cells (n = 9). For CDZ + ionomycin, [Ca2+]i = 0.23 ± 0.04 µM and dye transferred to 19.8 ± 0.7 cells (n = 6). For CDZ + ionomycin + 21.8 mM [Ca2+]o, [Ca2+]i = 0.81 ± 0.11 µM and dye transferred to 20.8 ± 0.7 cells (n = 5). **[Ca2+]i significantly higher than control or dye transfer significantly inhibited compared with control levels (P < 0.01).
) was added before and after Tg treatment to elicit Ca2+ release from intracellular stores. B: before incubation in nominally Ca2+-free buffer (in the absence of 250 nM Tg), dye transferred to 19.5 ± 0.6 cells (n = 4); 2–3 min after induction of capacitative Ca2+ entry, dye transfer was unchanged, with 19.8 ± 05 cells (n = 6) receiving dye.
