From the Department of Medical Biochemistry,
University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN,
United Kingdom and § Departamento de Investigacion, Servicio
de Neurologìa Experimental, Hospital "Ramon y Cajal,"
Carretera de Colmenar Km 9.1, 28034 Madrid, Spain
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ABSTRACT |
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Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by fusing the amino terminus of aequorin to the carboxyl terminus of connexins. The retention of function by the chimeric partners was investigated. Connexin 32-aequorin and connexin 43-aequorin retained chemiluminescent activity whereas that of connexin 26-aequorin was negligible. Immunofluorescent staining of COS-7 cells expressing the chimerae showed they were targeted to the plasma membrane. Gap junction intercellular channel formation by the chimerae alone and in combination with wild-type connexins was investigated. Stable HeLa cells expressing connexin 43-aequorin were functional, as demonstrated by Lucifer yellow transfer. Pairs of Xenopus oocytes expressing connexin 43-aequorin were electrophysiologically coupled, but those expressing chimeric connexin 26 or 32 showed no detectable levels of coupling. The formation of heteromeric channels constructed of chimeric connexin 32 or connexin 43 and the respective wild-type connexins was inferred from the novel voltage gating properties of the junctional conductance. The results show that the preservation of function by each partner of the chimeric protein is dictated mainly by the nature of the connexin, especially the length of the cytoplasmic carboxyl-terminal domain. The aequorin partner of the connexin 43 chimera reported calcium levels in COS-7 cells in at least two different calcium environments.
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INTRODUCTION |
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Gap junction intercellular channels allow the direct movement between neighboring cells of ions and small molecules generally less than 1 kDa (1). These cell-cell junctions are generally believed to underpin diverse integrative events such as tissue growth and differentiation (2) and the co-ordination of cardiac and smooth muscle cells (3). Thirteen highly related rodent connexin gap junction proteins have been identified (4). Connexins traverse the lipid bilayer four times with the amino and carboxyl tails facing the cytoplasm; the third membrane traverse M3 is postulated to line the channel (5). The main determinant of the molecular mass of connexins is the length of the cytoplasmic carboxyl tail, with amino acid sequence variation confined mainly to the single intracellular loop and carboxyl tail. The two outer "gap" facing loops facilitate the recognition and docking of hexameric connexon hemichannels contributed by adjacent cells (6). The docking and tight adhesion of aligned connexons in the plasma membrane results in the insulated extension of two transmembrane channels across the intercellular gap and is the final act in the assembly of connexins into gap junctions.
Calcium plays a central role in cellular communication, as shown by its demonstration as a key cytoplasmic factor in the control of junctional communication with increased intracellular calcium levels reducing gap junctional communication (7) by inducing transitions between apparent open and closed configurations of the channel (8). Intracellular calcium oscillations have been shown to spread as waves across groups of attached glial (9) and endothelial cells (10). These intercellular calcium waves proceed mainly via gap junctions, but the candidate-signaling component allowing intercellular crosstalk may not be calcium but a more global messenger molecule such as inositol 1,4,5-trisphosphate (11). Calcium may also be a crucial environmental factor at intracellular sites such as the endoplasmic reticulum-Golgi environs, where the oligomerization of connexins into hexameric connexons is thought to occur. Many calcium-binding proteins, some with chaperone-like functions, are localized to this region of the secretory pathway and roles in the oligomerization of virally encoded proteins have been demonstrated (12, 13). The involvement of calcium in connexin oligomerization, a prelude to gap junction biogenesis, may be either direct or indirect and can involve a role for accessory proteins such as calmodulin. Calmodulin regulates junctional physiology in a variety of physiological systems (14), and calmodulin binding domains in connexins have been delineated (15). Thus, knowledge of the calcium levels in the vicinity of the gap junctions and their connexin/connexon precursors will help to elucidate its relevance in the control of cell to cell signaling through gap junctions.
The first application of aequorin as a calcium-sensitive reporter, demonstrating the importance of free ionized Ca2+ in the gating of gap junction channels, was in 1975 by Rose and Loewenstein (16). Since then, aequorin has been targeted to a number of organelles, e.g. mitochondria (17), endoplasmic reticulum (18, 19), nucleus (20), and the plasma membrane (21), where it has been used to report the subcellular calcium environment. In the present work, we have adopted a similar approach by attaching aequorin to the carboxyl tail of connexins 26, 32, and 43 targeted to gap junctions. We then have analyzed the expression and functionality of the chimeric products in mammalian cells and Xenopus oocytes (22). The results show that functional gap junction channels were constructed in mammalian and amphibian cells. Furthermore, measurement of chemiluminescent activity of the connexin 43-aequorin chimera in transfected COS-7 cells reported at least two different calcium environments.
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EXPERIMENTAL PROCEDURES |
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Materials-- Taq DNA polymerase, the in vitro transcription/translation system (TNT),1 restriction and DNA modifying enzymes, and the Profection calcium phosphate transfection system were from Promega. ECL immunoblotting detection system and [35S]methionine were from Amersham Life Science, Inc. Tissue culture medium and reagents were supplied by Life Technologies, Inc. and monoclonal antibodies to Cx32 and Cx43 by Chemicon International and Zymed Laboratories Inc.. Oligonucleotides were prepared on site by J. Hoy. All other reagents were supplied by Sigma unless otherwise stated.
Construction of Connexin-Aequorin Chimeric
cDNAs--
Aequorin was fused in frame to the COOH terminus of
Cx26 (23), Cx32 (24), and Cx43 (25) using a two-step PCR procedure (26)
(Fig. 1). The first step generated
cDNA products containing overlapping regions of 3-connexin minus
the stop codon and 5
-aequorin minus the start codon. The chimeric
cDNAs were produced by a second PCR reaction, using equimolar
amounts of the initial PCR products and the 5
-connexin primer and the
3
-aequorin primer. The primers used were as follows:
Cx32P1, T7 promoter and first 15 bp of Cx32 (5
-CAG CTA ATA
CGA CTC ACT ATA GGG AGA ATG AAC TGG ACA GGT); Cx32P2, first
15 bp of Aeq (-ATG) and last 15bp of CX32 (-TAA) (5
-TGA TGT AAG CTT
GAC GCA GGC TGA TCG GTC); Aeq32, reverse of Cx32P2; Cx26P1, T7 promoter and first 15 bp of
Cx26 (5
-CAG CTA ATA CGA CTC ACT ATA GGG AGA GGA TCC ATG
GAT TGG GGC ACA); Cx26P2, first 15bp Aeq (
ATG) and last 15 bp of CX26 (
TAA) (5
-CAG CGG ATC CTT AGA CTG GTC TTT T);
Aeq26, reverse of Cx26P2; Cx43P1, SP6
promoter and first 15 bp of Cx43 (5
-CAG CGA TTT AGG TGA CAC TAT AGA
GAT CTA GAA TGG GTG ACT GGA GT;)
Cx43P2, first 15 bp of Aeq (
ATG) and last 15 bp of CX43
(
TAA) (5
-TGA TGT AAG CTT GAC AAT CTC CAG GTC ATC); Aeq43,
reverse of Cx43P2; Aeq5, T7 promoter and first 15 bp of Aeq (5
-TAA TAC GAC TCA CTA TAG GGA GAA ATG GTC AAG
CTT ACA TGA GAC TTC GAC); Aeq10, last 15 bp of aequorin including stop codon (5
-CTC CTT GAG CTC GTC GAC TTA GGG
GAC AGC TCC AC). The external primers contain BamHI
(Cx26P1, Cx32P1) or BglII restriction
enzyme sites (Cx43P1, Aeq10) to enable cloning and characterization of clones. The chimeric PCR products were cloned
directly into a TA cloning plasmid, PCR3 (Invitrogen). This plasmid has
a T7 promoter and cytomegalovirus immediate early promoter, thus
allowing analysis of expression both in vitro and in
vivo. Positive clones were identified by miniplasmid preparations and restriction enzyme analysis (27). The selected constructs were
sequenced using the PRISM dye terminator cycle sequencing kit
(Perkin-Elmer), and the results confirmed that no mutations were
present.
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Analysis of Aequorin Activity-- For analysis in vitro, several clones were selected for each chimera. Proteins expressed in a TNT system were analyzed on 12.5% SDS-polyacrylamide gels (28) and were fixed, enhanced, and vacuum-dried prior to autoradiography. Aliquots of TNT product (10 µl) were reconstituted in a buffer containing 500 mM NaCl, 1 µM coelenterazine, 1 mM EDTA, 20 mM Tris, pH 7.4. Calcium-sensitive chemiluminescent activity of proteins was analyzed in a luminometer (29, 30).
For analysis in vivo, COS-7 cells (4 × 105 cells), in six-well dishes, were transfected with 5 µg of the relevant plasmid DNA using calcium phosphate precipitation. Cells were harvested 48 h after transfection in 500 µl of hypo-osmotic buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 5 mMMeasurement of Intracellular Calcium-- For the aequorin-related Ca2+ measurements, COS-7 cells were transfected with 5 µg of Cx43-Aeq DNA, or were infected with a replication-deficient adenovirus vector expressing a cytosolic luciferase-aequorin chimera (31). Twenty-four hours later, the cells were detached in cold phosphate-buffered saline (120 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·2H2O, pH 7.4), containing 1 mM EDTA, and seeded onto glass coverslips. To increase expression of the recombinant proteins, 5 mM sodium butyrate was included in the culture medium for 18 h prior to the experiments (32). Forty-eight hours after transfection, the recombinant apoaequorins were converted into photoproteins by addition of coelenterazine (final concentration 5 µM) for 4 h at 37 °C before experiments. In experiments in which aequorin was used to measure free Ca2+ under the plasma membrane, activation with coelenterazine was conducted in the presence of 1 mM EGTA (21). Coverslips were inverted over the reservoir of a plastic perfusion chamber (maintained at 37 °C in a dark box) and brought into contact with a fiber optics bundle attached to a photon counting camera (Photek three-microchannel plate intensified CCD camera (Photek 216)) (33). Cells were perfused for at least 10 min in KRH (120 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1 mM EDTA, 25 mM Hepes, pH 7.4, 1.3 mM CaCl2; for subplasma membrane measurements, CaCl2 was substituted by 1 mM EGTA) to remove excess coelenterazine. The fractional discharge of aequorin was calculated from the total light emitted by the photoprotein at the end of each experiment, by exposing the cells to water containing 5 mM CaCl2 to discharge unconsumed aequorin. This value was used to convert light emission into free Ca2+ (34). Identical calibration curves for aequorin and Cx43-Aeq were obtained as described in Ref. 35.
In Vivo Expression and Cellular Localization-- For Western blot analysis, 100-mm dishes containing COS-7 cells were transfected with 20 µg of the relevant cDNA. Forty-eight hours after transfection, cells were harvested in 500 µl of 40 mM Tris, pH 7.4, 1 mM EDTA containing protease inhibitors (5 µg/ml each leupeptin, aprotinin, antipain, and chymostatin and 0.5 µg/ml pepstatin A) and freshly prepared phenylmethylsulfonyl fluoride (1 mM), left for 1 min at room temperature, and homogenized through a 16-mm gauge needle followed by brief sonication. Cells were centrifuged for 15 min at 500 × g to remove nuclei, and the supernatant was then centrifuged at 25,000 × g for 1 h at 4 °C. Cell pellets were resuspended in 30 µl of a solubilizing buffer (28). Aliquots (10 µl) were analyzed on 12.5% SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis. Proteins were characterized using a primary rabbit anti-aequorin antibody (31) and secondary horse radish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad). Blots were developed using the enhanced chemiluminescence (ECL) system.
For immunolocalization, COS-7 cells were plated onto 16-mm2 coverslips in 12-well dishes and transfected with 5 µg of the relevant cDNA. Cells were fixed 48 h after transfection in 4% formaldehyde and permeabilized in 0.1% Triton X-100, 0.1 M lysine in phosphate-buffered saline. The cellular localization of the chimeric proteins was determined using a monoclonal Cx32 antibody (generated to a sequence on the intracellular loop) or a monoclonal Cx43 antibody (generated to a sequence on the carboxyl tail). A rabbit polyclonal antibody to the intracellular loop of Cx26 was also used (36). Aequorin was localized using a rabbit anti-aequorin antibody (31).Functional Analysis of Chimeric Cx-Aeq in Stable Cell
Lines--
HeLa cells were transfected with 20 µg of relevant Cx-Aeq
DNA in the PCR3 plasmid as described above. Forty-eight hours after transfection, the cells were split into selective media (Dulbecco's modified Eagle's medium containing 400 µg/ml G418 sulfate) and clonally propagated (37). Clones were selected on the basis of their
aequorin activity. The ability to form functional gap junction channels
was assessed by microinjection of confluent cell monolayers with 5%
w/v Lucifer yellow CH in 0.3 M LiCl, followed by
examination by fluorescence microscopy using a 440-nm filter. The cells
were then fixed in 4% formaldehyde for 30 min, mounted under
16-mm2 coverslips, and the number of injected cells
transferring dye counted. In control experiments, dye transfer was
assessed in cells incubated with 18-glycyrrhetinic acid (1 µM) for 30 min, a potent blocker of gap junction-mediated
intercellular communication (38).
Functional Analysis of Chimeric Cx-Aeq in Xenopus
Oocytes--
To express the chimerae in Xenopus oocytes,
each wild-type and chimeric cDNA was transferred into the
BglII site of the Melton-derived plasmid pBSKXG (39). This
flanked the chimeric DNA inserts with the 5- and 3
-untranslated
region of the Xenopus
-globin gene to improve the
efficiency of translation in oocytes.
Preparation of cRNAs--
Wild-type and chimeric Cx-Aeq
cDNAs were linearized with ClaI and sense transcribed
with T7 polymerase in the presence of the cap analogue m7G(5)ppp(5
)G
(Boehringer Mannheim). After DNase digestion and purification, cRNAs
were quantified by absorbance (260 nm) and the proportion of
full-length transcripts (>95%) was checked in 1% agarose gels
stained with ethidium bromide.
Expression in Pairs of Xenopus Oocytes-- Oocytes were prepared for injection as described previously (40) and co-injected with an antisense oligonucleotide (10 ng/oocyte) directed against Xenopus Cx38 mRNA to block endogenous expression (40) and the cRNAs (0.1-1.0 µg/µl; 50 nl/oocyte) encoding the chimeric and wild-type connexins. Vitelline membranes were removed in hypertonic solution 24 h after injection, and pairs of oocytes were placed in contact (22).
Measurement of Macroscopic Junctional
Conductance--
Junctional conductance developed between oocyte pairs
was recorded 24-72 h after pairing using the dual voltage clamp
technique with two independent amplifiers (TEV-200, Dagan). Each cell
of the pair was impaled with two microelectrodes (0.5-1 megohm) filled with 2 M KCl, 10 mM EGTA, and 10 mM
Hepes pH 7.2. Junctional conductance was measured as follows;
V1, I1,
V2, and I2 are voltages
and currents, with the holding current subtracted, in oocyte 1 and 2, respectively. Initially both oocytes were clamped at the same holding
potential (Vm, e.g. 40 mV), when the
transjunctional voltage (Vj) was zero since
Vj = V1
V2. A voltage step, applied in oocyte 1, defined
positive Vj as greater relative positivity in oocyte
1. The current injected in oocyte 2, I2, i.e. to hold its potential constant, was equal in magnitude
and opposite in sign to the current flowing through the junctional channels (Ij =
I2). Thus,
the macroscopic junctional conductance was directly calculated as
Ij/Vj. The stimulation and data
collection were carried out with a PC-AT computer using pCLAMP software
and Digidata 1200-A interface (Axon Instrument Inc.).
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RESULTS |
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In Vitro Expression of Chimeric Connexins-- The chimeric proteins were initially characterized after TNT to determine their molecular mass and whether functionality of the aequorin partner was retained. Chimeric proteins of the predicted size were synthesized from the three chimeric constructs, namely Cx26-Aeq (47 kDa), Cx32-Aeq (53 kDa), and Cx43-Aeq (64 kDa) (data not shown). Aequorin activity of Cx43-Aeq and Cx32-Aeq was comparable to that of wild-type aequorin, but chemiluminescence of Cx26-Aeq chimera was markedly reduced. The effect of fusion of aequorin to Cx26 on its chemiluminescence was investigated by PCR amplification of the chimera and the aequorin partner of each chimera. The PCR products were analyzed for aequorin activity in the TNT translation assay. The aequorin partner amplified from Cx26-Aeq gave comparable aequorin activity (62 ± 1.5%) to Cx32-Aeq (78 ± 6.4%) and Aeq-WT (100%). However, chemiluminescence obtained with the full-length Cx26-Aeq construct was only 1.3 ± 0.1% of Aeq-WT activity. These results show that fusion of aequorin to the carboxyl terminus of connexin 26 inhibited aequorin activity in vitro.
In Vivo Analysis of Chimeric Connexins-- Indirect immunofluorescence of transfected COS-7 cells indicated that Cx32-Aeq and Cx43-Aeq chimerae were targeted to the plasma membrane (Fig. 2a). Furthermore, double immunofluorescence with aequorin and connexin antibodies showed that aequorin was localized to the same region of the cell as its connexin partner (data not shown). Thus, the results demonstrated that the two chimeric proteins were located at the plasma membrane and at intracellular locations, especially in the endoplasmic reticulum-Golgi environs of the cell, as established by immunostaining with antibodies to TGN38, a Golgi marker (41) (data not shown). Although Cx26 was localized by immunocytochemistry using an intracellular loop domain antibody (Des 3) (36), co-localization of the connexin and aequorin components of Cx26-Aeq could not be demonstrated since no signal was detected with aequorin antibodies.
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Formation of Cell-Cell Channels in HeLa Cell Transfectants--
To
study the formation of functional gap junction channels, stable HeLa
cell lines expressing the chimeric proteins were generated and their
intercellular communication properties assessed by Lucifer yellow
transfer (Fig. 3 and Table
II). Fig. 3 (A1 and
A2) shows that two transfected HeLa cell lines expressing
Cx43-Aeq (D7 and E9) transferred dye to up to 25 neighboring cells,
whereas negligible dye transfer occurred in nontransfected cells (Fig.
3, B1). Confirmation that direct communication had occurred
between cells across gap junction channels was obtained by inhibition
of dye transfer following incubation of the cells for 30 min with 18
glycyrrhetinic acid, an inhibitor of intercellular communication across
gap junctions (Fig. 3, A3 and B2). Additionally,
a correlation was noted between dye transfer and the level of aequorin
expression (Table II). In HeLa cell line D7, which showed the higher
aequorin activity, 74% of the cells injected with Lucifer yellow
transferred dye to 10 or more cells. In cell line E9, which exhibited
significantly lower aequorin activity, 59% of the cells transferred
the dye to the same number of cells. Few (<2%) nontransfected HeLa
cells transferred dye to neighboring cells. These results indicate that the expression properties of the two partners of the chimeric protein
are closely linked. Thus, HeLa cells expressing Cx32-Aeq transferred
the dye only when protein expression from the cytomegalovirus immediate
early promoter was stimulated by 5 mM sodium butyrate. No
intercellular dye transfer was observed between HeLa cells expressing
Cx26-Aeq.
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Formation of Cell-to-Cell Channels in Xenopus Oocytes-- The voltage gating properties of the channels formed by Cx43-Aeq and Cx32-Aeq were examined. To study the assembly of the chimerae into functional gap junctions alone (i.e. as homomeric channels) or in combination with wild-type connexins (i.e. as heterotypic and heteromeric channels), paired oocytes were injected with various combinations of connexin and Cx-Aeq cRNAs. Homomeric and heterotypic channel formation was determined by measuring the macroscopic junctional conductance. Formation of heteromeric channels was inferred by comparing the voltage gating properties of the junctions formed in pairs of oocytes expressing only wild-type connexins and the results obtained with pairs co-expressing the chimerae.
Homomeric and Heterotypic Channels Comprising Cx-Aeq Subunits-- To examine the formation of homotypic channels, paired oocytes were injected with equal amounts of the same connexin-aequorin chimera cRNA (Table III, a and b). Although large junctional conductances were induced by cRNA of wild-type connexins, pairs expressing Cx26-Aeq or Cx32-Aeq showed no detectable levels of electrical coupling. In contrast, pairs injected with Cx43-Aeq were electrically coupled, although junctional conductance was 1-2% of that of wild-type Cx43, despite the fact that both pairs expressed similar levels of connexins. These experiments show that expression of Cx26-Aeq and Cx32-Aeq in oocytes does not result in the formation of intercellular channels, whereas the Cx43-Aeq was able to form channels, although this chimera was far less effective than Cx43 in inducing homomeric coupling.
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Voltage-gating Properties of Cx43-Aeq Channels-- Many vertebrate junctions are controlled by transjunctional voltage, i.e. the voltage difference between cell interiors (Vj), in which voltage-gating properties are specific depending on the type of connexin forming the intercellular channels (42). The voltage gating properties of intercellular channels comprising Cx43-Aeq subunits showed that junctional conductances (gj) of homotypic Cx43-Aeq and Cx43 junctions displayed a common maximal gj at Vj = 0 and symmetrical gj reduction in response to positive and negative Vj (Fig. 4). However, they possessed different kinetic properties since the gj transitions of Cx43-Aeq junctions showed markedly slower time courses.
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Formation of Heteromeric Cx/Cx-Aeq Channels-- The formation of heteromeric channels comprising chimeric Cx-Aeq and wild-type connexin and the effect of chimeric Cx26, Cx32, and Cx43-Aeq on the coupling induced by cells expressing wild-type connexins were studied (Table III, d, e, and f). Pairs co-expressing Cx32 and Cx32-Aeq in both oocytes developed significantly lower levels of coupling than those expressing solely the wild-type connexin. This reduction of junctional conductance was less in pairs in which one cell was co-injected with cRNA to Cx32-Aeq and the other with cRNA to Cx32. This dominant-negative inhibition may be explained by oligomerization of chimeric and Cx32 subunits. Support for this conclusion was obtained by comparing the voltage gating properties of junctional conductance developed between pairs expressing only wild-type Cx32 with those formed by co-injecting one cell with the cRNAs to Cx32 and Cx32-Aeq with the counterpart oocyte being injected solely with cRNA to Cx32 (Fig. 5). In contrast to wild-type Cx32 junctions, where conductance responded symmetrically to positive and negative pulses of Vj conductance (40), the junctions in Cx32/Cx32-Aeq:Cx32 pairs reacted asymmetrically to Vj steps of opposite polarity. This asymmetry strongly suggests heterogeneity in the connexin composition of the connexon hemichannels contributed by each cell. Since Cx32-Aeq was unable to form functional channels alone or in heterotypic combination with the wild-type hemichannels described above, this result also points to the co-oligomerization of chimeric and wild-type Cx32 subunits into heteromeric connexon hemichannels. These heterotypic junctions showed complex voltage regulation because the incorporation of chimeric Cx32-Aeq subunits into junctional channels only slightly modified the voltage gating properties of heteromeric Cx32-Aeq/Cx32 hemichannels. There was also a more dramatic reduction in voltage sensitivity and a slowing of the junctional conductance inactivation of the apposed Cx32 homomeric hemichannels. It was found that Vj positive in the cytoplasmic side of the oocyte expressing both Cx32 and Cx32-Aeq induced junctional currents that were less voltage-sensitive and that displayed slower kinetics than those induced by negative Vj, as was observed in the homotypic Cx32 junctions.
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Utility of Cx-Aeq Chimerae for Monitoring Calcium Levels-- COS-7 cells expressing Cx43-Aeq were used to measure by chemiluminescence the intracellular calcium concentration [Ca2+]i. The basal calcium reported by Cx43-Aeq was approximately 415 nM compared with 100 nM reported by luciferase-aequorin used as a control for cytosolic calcium concentrations (31) (Fig. 6A). When aequorin was reconstituted in situ in cells exposed to calcium-deficient medium containing EGTA, and calcium was then added back to the medium, a transient increase in the [Ca2+]i, reaching a maximum of 2 µM, was observed. This reflected a calcium signal emanating from the under-surface of the plasma membrane (Fig. 6B). Since Cx43-Aeq was located immunocytochemically in at least two locations within the cell, namely in the endoplasmic reticulum- Golgi environs and at the plasma membrane (Fig. 2a), these results suggest that the basal calcium concentration reported by Cx43-Aeq is a combination of the high [Ca2+]i at the plasma membrane and that in microdomains located along the secretory pathway. Alternatively, the [Ca2+]i monitored in the cytosol may reflect specific microdomains of high [Ca2+]i where the Cx43-Aeq intracellular stores reside.
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DISCUSSION |
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The Assembly of Chimeric Proteins into Functional Gap Junctions-- The chimerae examined in this work comprise a targeting component (connexin) and a reporter group (aequorin). During the assembly of functional gap junction channels from connexins with attached reporter groups, each partner should retain biological activity. The reporter group is required to function irrespective of its position in the cell, but the functionality of the targeting component can only be assessed following its assembly into connexon hemichannels that align and interact at the plasma membrane with connexons in neighboring cells to generate a gap junction. The chimerae should oligomerize and traffic along the secretory pathway with fidelity. At the plasma membrane, the hemichannels should dock subject to the connexin compatibility rules determined in studies on gap junction formation in HeLa cells (43) and in Xenopus oocytes (44). A further objective is that the gap junctions should operate as near to normal as possible, although the main task of the chimera is to report the calcium environment en route to and at its functional residence. The present work shows that Cx43-Aeq best satisfied these demanding criteria; Cx43-Aeq was targeted to the gap junctions as shown by Lucifer yellow transfer in HeLa cells and maintained the ability to form homotypic channels in oocytes. The chimeric Cx43-Aeq subunits induced low levels of macroscopic junctional conductance relative to wild-type connexin. Finally, the utility of Cx43-Aeq was evident for it reported the calcium environment in at least two different Ca2+ environments in live cells.
The Cx32-Aeq chimera showed characteristics similar to other chimeric connexins that have been studied (45) as well as to mutants of human Cx32 associated with the X-linked form of Charcot-Marie-Tooth disease (46). These modified connexons combine the loss of ability to form homomeric channels with the effect of dominant-negative inhibition over the development of coupling induced by wild-type connexins. However, in the present work, Cx32-Aeq also incorporated together with wild-type subunits into electrophysiologically distinguishable heteromeric channels, indicating that oligomerization with a functionally competent connexin partner rescued the complete loss of function of Cx32-Aeq. The ability to form a functional heterotypic channel may be critically determined by the stoichiometry of Cx32-Aeq and wild-type subunits in the connexon hemichannels and a number of different hemichannels could result from different combinations of wild-type and Cx32-Aeq subunits. The assembly of two types of subunits into heteromeric hemichannels may inhibit the correct alignment of the two participating hemichannels, the docking, or the gating of the complete channel into the open configuration. The results also suggest that only a few classes of the multiple possible combinations, (probably those with lowest Cx32-Aeq:Cx32 ratio), resulted in the formation of functional heteromeric channels. In contrast to results obtained with Cx32-Aeq and Cx43-Aeq, the Cx26-Aeq chimera was unable to incorporate into functional gap junction channels. The position where the reporter is attached to the connexin is important. The options are the amino terminus, the intracellular loop and the carboxyl tail, all cytoplasmically located (4). The amino terminus (~22 amino acids) shows high sequence homology in all connexins, suggesting a crucial function, especially in membrane insertion and possibly targeting (47). The intracellular loop, highly variable in sequence, is implicated in channel regulation (48). The carboxyl-terminal tail was chosen in the present work for it may position the aequorin reporter group further away from the membrane, thereby optimizing retention of functions of both partners. Furthermore, functional analysis of a truncated Cx43, in which most of the carboxyl-terminal domain was removed, has suggested that the cytoplasmic tail does not play an important role in transjunctional voltage gating (49). However, in the absence of a detailed three-dimensional structure, the proximity and interactive dynamics of these domains remain to be investigated. Connexins 26, 32, and 43 have cytoplasmic carboxyl domains of 18, 78, and 156 amino acids respectively (3). The present work shows that Cx26-Aeq was not functional, with even the aequorin activity impaired. Cx32-Aeq alone was also defective in its ability to form channels in oocytes although the chimera's topography in the membrane was correct and dye transfer occurred in transfected HeLa cells upon induction of protein expression with sodium butyrate. In oocytes, electrical communication was absent but was rescued if the chimera was incorporated into heteromeric channels with Cx32. These observations, combined with the fact that Cx43-Aeq formed functional homomeric channels both in oocytes and in HeLa cells, lead to the view that the length of the carboxyl tail emerges as one of the most important criteria in maintaining optimal aequorin expression. We conclude that the ratio of the molecular weight of connexin to its partner protein is likely to be important in maintaining optimal functionality of both partners of the chimeric protein. For example, fusion of the large reporter molecule,Chimeric Connexins Modify Gap Junction Channel Operation-- The basis by which gap junction channels are gated by voltage, intracellular Ca2+, or pH acidification remains unresolved. Calcium has been suggested to trigger a twisting action of connexins leading to pore closure at the cytoplasmic side of the junction (8). Gating by pH may involve a "particle-receptor" interaction between the intracellular loop and the carboxyl terminus of Cx43 leading to closure of the gap junction (51). Several connexin domains have been implicated in the gating involved in transjunctional voltage dependence. Point mutations in Cx32 and Cx26, which alter the charge of the NH2-terminal second amino acid and at the M1/E1 boundary, reversed gating polarity (52). The substitution of Pro87 by Leu in Cx26 suggests that the second membrane traverse (M2) is active in the transduction event in voltage gating (53), and evidence for participation of the cytoplasmic loop in voltage gating has also been reported (46). Clearly, attachment of a large molecule of similar overall size such as aequorin to the carboxyl tail of a connexin can modify any of the gating mechanisms in junctional channels. Indeed, as was shown by the novel gating properties displayed by Cx43-Aeq homotypic and Cx32-Aeq heterotypic channels, at least the voltage gating mechanism was altered in oocytes. However, it should be noted that, despite these differences, gap junctions constructed of Cx43-Aeq in transfected HeLa cell lines actively transferred Lucifer yellow. Thus, mammalian cells and oocytes behave differently with respect to their handling and expression of connexin chimerae and their assembly into connexon channels. These observations support the view that eukaryotic cells and oocytes can display different gap junction communication characteristics (54).
Cx43-Aeq Reports Intracellular Calcium Levels in COS Cells-- Finally, we show that the chimeric connexin probes can be used to report [Ca2+]i in live cells. Basal [Ca2+]i reported by Cx43-Aeq was approximately 415 nM. However, after the aequorin moiety of the chimera had been reactivated in medium containing EGTA, a transient level reaching 2 µM was reported. These results show that two different components of intracellular [Ca2+]i can be reported by the same probe. The transient (2 µM) calcium signal may reflect store-operated calcium influx (55). However, this value (2 µM) is similar to that obtained using a SNAP25-Aeq chimera targeted exclusively to the plasma membrane (21). The basal Ca2+ level reported by Cx43-Aeq was higher than that reported by the cytosolically located luciferase-aequorin probe (approximately 100 nM). This elevated [Ca2+]i may reflect a mean of the [Ca2+]i in the endoplasmic reticulum-Golgi areas where the intracellular stores of Cx43-Aeq reside, and the high level reported below the plasma membrane. However, since the results obtained with oocytes and HeLa cells show that Cx43-Aeq was targeted to the plasma membrane and that gap junction mediated intercellular communication occurred, it is also possible that the chimera is reporting the calcium level near to the gap junction pore entrance. The utility of these functional connexin-aequorin probes will allow many areas of work impinging on gap junction biogenesis and gating to be explored.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. V. Gire (University of Wales College of Medicine) for assistance with microinjection experiments.
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FOOTNOTES |
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* This work was supported by Medical Research Council Grant G-9305117 (to W. H. E.) and by Fondo de Investigaciones Sanitarias Grant 95/0643 (to L. C. B.).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.
¶ To whom correspondence should be addressed. E-mail: wmbwhe{at}cardiff.ac.uk.
1 The abbreviations used are: TNT, coupled in vitro transcription and translation; Cx, connexin; Aeq, aequorin; Cx26-Aeq, connexin 26-aequorin chimera; Cx32-Aeq, connexin 32-aequorin chimera; Cx43-Aeq, connexin 43-aequorin chimera; Aeq-WT, wild-type aequorin; V, voltage; I, current; Vm, holding potential; Vj, transjunctional voltage; gj, junctional conductance; PCR, polymerase chain reaction; [Ca2+]i, intracellular calcium concentration; bp, base pair(s).
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REFERENCES |
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