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First published online May 10, 2006
doi: 10.1242/10.1242/jcs.02940
Research Article |
3 connexin prevents severe cataracts caused by an 8 point mutation
1 School of Optometry and Vision Science Program, University of California at Berkeley, Berkeley, CA, USA
2 Program in Genetics, State University of New York, Stony Brook, NY, USA
3 The Jackson Laboratory, Bar Harbor, ME, USA
4 Department of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta, GA, USA
5 Department of Physiology and Biophysics, State University of New York, Stony Brook, NY, USA
* Author for correspondence (e-mail: xgong{at}berkeley.edu)
Accepted 15 February 2006
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Summary |
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8 connexin (Cx50) has been previously shown to cause a severe cataract by interacting with endogenous wild-type 3 connexin (Cx46) in mouse lenses. Here, we tested whether a knocked-in 3 connexin expressed on the locus of the endogenous 8 connexin could modulate the severe cataract caused by the 8-G22R mutation. We found that the 3(-/-) 8(G22R/-) mice developed severe cataracts with disrupted inner fibers and posterior rupture while the 3(-/-) 8(G22R/KI3) lens contained relatively normal inner fibers without lens posterior rupture. The 8-G22R mutant proteins produced typical punctate staining of gap junctions between fiber cells of 3(-/-) 8(G22R/KI3) lenses, but not in those of 3(-/-) 8(G22R/-) lenses. Thus, we hypothesize that the knocked-in 3 connexin subunits interact with the 8-G22R connexin subunits to form functional gap junction channels and rescue the lens phenotype. Using an electrical coupling assay consisting of paired Xenopus oocytes, we demonstrated that only co-expression of mutant 8-G22R and wild-type 3 connexin subunits forms functional gap junction channels with reduced conductance and altered voltage sensitivity compared with the channels formed by 3 connexin subunits alone. Thus, knocked-in 3 connexin and mutant 8-G22R connexin probably form heteromeric gap junction channels that influence lens homeostasis and lens transparency.
Key words: Gap junction, Connexin, Knock-in 3 (KI3), Cataract
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Introduction |
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The ocular lens is an avascular organ that continually grows throughout life and must remain transparent to transmit light images onto the retina. Gap junction channels play important roles in lens homeostasis by providing low-resistance pathways for both electrical and metabolic coupling of lens cells. Lens gap junction channels are formed by three connexins that have distinct and redundant expression. The 1 connexin gene (known as Gja1, and also as Cx43 and connexin 43) is specifically expressed in lens epithelial cells, the 3 connexin gene (known as Gja3, and also as Cx46 and connexin 46) is mainly expressed in lens fiber cells, and the 8 connexin gene (known as Gja8, and also as Cx50 and connexin 50) is expressed in both epithelial cells and fiber cells. Genetic studies have demonstrated the importance of gap junction channels formed by 3 and 8 connexins in maintaining lens transparency and proper lens growth, respectively. Deletion of the 3 connexin resulted in lens nuclear cataracts in mice (Gong et al., 1997
), while the knockout of 8 connexin caused smaller lenses with mild opacities (White et al., 1998; Rong et al., 2002). In addition, genetic replacement of endogenous 8 connexin with wild-type 3 connexin by homologous recombination (homozygous 3 connexin knock-in mice, hereafter referred to as KI3 mice) prevented lens opacity but did not revert the reduction in lens size caused by the absence of 8 (White, 2002). Further studies demonstrated that the knocked-in wild-type 3 connexin expressed from the 8 locus, but not from the endogenous 3 locus, was sufficient to maintain lens transparency (Martinez-Wittinghan et al., 2003).
The Lop10 mutant mice developed autosomal semi-dominant cataracts (Runge et al., 1992); further studies identified a substitution of Gly to Arg at codon 22 of 8 connexin (8-G22R) in these mice (Chang et al., 2002). Hereafter, the Lop10 mutant mice are referred to as 8(G22R/G22R) mice. Homozygous 8(G22R/G22R) mice developed microphthalmia with dense cataracts resulting from severely disrupted cortical fibers with vacuoles and ruptured posterior lens capsule. Reduced levels of phosphorylated forms of endogenous wild-type 3 subunits were detected in lenses of 8(G22R/G22R) mice. Double homozygous 3(-/-) 8(G22R/G22R) mice developed cataracts with relatively normal peripheral fibers (Chang et al., 2002). Thus, the presence of endogenous 3 connexin seems to increase the severity of cataract caused by the 8-G22R mutation.
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8-G22R subunits directly interact with wild-type 3 subunits to influence lens homeostasis by altering gap junction formation and channel properties, which modulates cataract formation. To test this hypothesis and to examine the anti-cataract effect of KI3, we have generated mutant mice that express both mutant 8-G22R connexin and knocked-in 3 connexin and studied the morphological and biochemical properties of mutant lenses. We have found that lens phenotypes are affected by the combination of mutant 8-G22R and knocked-in 3 connexins in vivo and have determined unique channel properties of gap junctions formed by wild-type 3 and 8-G22R mutant subunits in vitro. |
Results |
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3 connexin prevents lens rupture and substantially reduces cataract caused by the 8-G22R mutation3 connexin modulates the lens phenotypes caused by the 8-G22R mutation in vivo, we generated and characterized three different compound mutant mice: 3(-/-) 8(G22R/-), 3(-/-) 8(KI3/-) and 3(-/-) 8(G22R/KI3). These mice were produced by intercrossing 3(-/-) 8(G22R/-) with 3(-/-) 8(KI3/-) animals. Since these mice were in the 3(-/-) background, we eliminated the contribution of endogenous wild-type 3 connexin in their lenses. The 3(-/-) 8(KI3/-) mice developed clear lenses (Fig. 1A), similar to previously described lenses in the knockover 3(-/-) 8(KI3/KI3) mice (Martinez-Wittinghan et al., 2003). By contrast, the 3(-/-) 8(G22R/-) mice developed severe cataracts and lens posterior rupture (Fig. 1B). Interestingly, the 3(-/-) 8(G22R/KI3) mice developed lenses with only very mild nuclear cataracts, and no posterior rupture (Fig. 1C). A summary of lens phenotypes of different connexin mutant mice is listed in Table 1. Representative histological data of mice at postnatal day 14 showed that the 3(-/-) 8(G22R/-) lenses developed severely altered inner fibers, fiber degeneration and lens posterior rupture (Fig. 2A); however, the 3(-/-) 8(G22R/KI3) lenses had relatively normal fiber cells without lens posterior rupture (Fig. 2B). Thus, unlike the endogenous wild-type 3 connexin, the knocked-in 3 subunits suppressed the severe cataract and prevented lens posterior rupture that occurred in the 3(-/-) 8(G22R/-) lenses.
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Knocked-in 3 connexin stabilizes protein expression and restores typical gap junctions containing mutant 8-G22R subunits in the lens fiber cells
To understand the underlying molecular mechanism as to how knocked-in 3 connexin subunits rescued the lens phenotype, we characterized the distribution and expression of mutant 8-G22R connexin subunits in lenses of 3(-/-) 8(G22R/-) and 3(-/-) 8(G22R/KI3) mice by immunostaining and immunoblotting, using a polyclonal 8 antibody that was previously shown to recognize the 8-G22R subunits (Chang et al., 2002). We first examined the subcellular distribution of 8-G22R subunits in frozen lens sections of 3(-/-) 8(G22R/KI3) and 3(-/-) 8(G22R/-) mice at the age of 3 weeks. Only weak fluorescent signals without a typical junctional staining pattern corresponding to expression of the 8-G22R protein were found in the outermost fiber cells of lenses of 3(-/-) 8(G22R/-) mice (Fig. 3A, 3C and C'). By contrast, much stronger 8-G22R connexin signals, with a typical punctate staining pattern, were predominantly located in the long sides of both the peripheral and inner lens fiber cells of lenses of 3(-/-) 8(G22R/KI3) mice (Fig. 3B, 3D and D'). Consistent with the immunostaining data, western blotting further confirmed that the 8-G22R protein was poorly expressed in the membrane-enriched fraction of lenses from 3(-/-) 8(G22R/-) mice, but was obviously present in a much higher amount in the equally loaded membrane-enriched fraction isolated from lenses of 3(-/-) of 8(G22R/KI3) mice (Fig. 4). Moreover, immunoblotting showed that protein levels of knocked-in 3 connexin remained largely unchanged in lenses of 3(-/-) 8(G22R/KI3) mice compared with those of 3(-/-) 8(KI3/-) mice. Thus, the presence of knocked-in 3 connexin in the lens restored or stabilized the expression of mutant 8-G22R subunits and targeted them to fiber cell membranes.
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Transmission electron microscopy results verify the presence of gap junctions between the fiber cells of 3(-/-) 8(G22R/KI3) lenses
To determine whether mixing knocked-in 3 and 8-G22R affected gap junction plaques in lens fibers, we used ultra-thin lens sections and transmission electron microscopic (TEM) analysis to examine the three-compound mutant lenses. TEM data showed that 16-17 nm thick gap junctions formed in the 3(-/-) 8(KI3/-) lens fibers (Fig. 5A and A'), whose channels were presumably formed by knocked-in 3 connexin subunits only. By contrast, only loosely packed membranes of adjacent fiber cells without gap junctions were observed in the 3(-/-) 8(G22R/-) lens (Fig. 5B and B'). Typical pentalamellar gap junction structures were also detected between the fiber cells of the 3(-/-) 8(G22R/KI3) lens (Fig. 5C and C'). These gap junctions were composed of either knocked-in 3 connexin subunits alone, or a mixture of knocked-in 3 and 8-G22R subunits. The TEM data demonstrated that the 8-G22R subunits alone were unable to form gap junctions in the lens fiber cells, and further suggested that the stabilization and targeting of the 8-G22R subunits by knocked-in 3 connexin corresponds to the incorporation of 8-G22R into gap junction plaques containing both subunits.
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3 and 8-G22R subunits produces heteromeric gap junction channels in vitro3 subunits rescue the lens phenotype by heterotypically and/or heteromerically interacting with 8-G22R subunits to form gap junction channels. To test this hypothesis, we examined electrical coupling properties between paired Xenopus oocytes expressing 8-G22R subunits with or without 3 subunits. Immunoblotting verified that wild-type 3 connexin or 8-G22R subunits were efficiently translated in oocytes injected with wild-type 3 connexin or 8-G22R cRNAs, and that oocytes co-injected with both cRNAs expressed equivalent amounts of 3 and 8-G22R subunits as the single injections (Fig. 6A). We then measured the gap junctional conductance of oocyte pairs injected with various cRNA combinations of wild-type 3 and 8-G22R connexins (Fig. 6B). Control oocyte pairs injected with water showed negligible junctional conductance. As expected, wild-type 3 cRNA-injected pairs exhibited high levels of conductance with a mean value of 
19 µS. By contrast, the 8-G22R cRNA-injected pairs had insignificant levels of gap junctional conductance, indistinguishable from the values measured in the water-injected negative-control pairs. Heterotypic oocyte pairs that had one cell injected with wild-type 3 and the other with 8-G22R cRNAs also failed to develop gap junctional conductance above the negative-control value. However, when the wild-type 3 and 8-G22R cRNAs were co-injected into the same oocyte, the resulting heteromeric channels developed gap junctional conductance with a mean value of 5 µS, approximately 75% lower than the junctional conductance formed by wild-type 3 alone. This reduction in coupling was statistically significant (P<0.05) and occurred despite equal levels of 3 protein synthesis in the two types of oocyte pairs (Fig. 6A). Since the levels of 3 expression were identical, one explanation for the 75% reduction in conductance is, when 3 and 8-G22R are co-injected, the formation of heteromeric channels. These data also demonstrate that mutant 8-G22R subunits could not make homotypic channels by themselves; nor could they form heterotypic channels with cells expressing wild-type 3 subunits.
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Heteromeric 3 and 8-G22R gap junction channels have altered voltage-gating properties
If the co-injected 8-G22R and 3 subunits interact to form heteromeric channels, the gating properties of the junctional conductance might be distinct from those of homotypic 3 channels. To test this possibility, we examined the kinetics and equilibrium properties of gap-junction voltage dependence in wild-type 3-injected, and 8-G22R- and 3-co-injected oocyte pairs. When cell pairs expressing homotypic wild-type 3 channels were subjected to a series of transjunctional voltages (±120 mV in 20 mV steps), the junctional currents declined slowly at values >40 mV. In co-injected 8-G22R and 3 heteromeric pairs, the junctional currents declined more rapidly and at lower values of transjunctional voltage (> 20 mV, Fig. 7A). Both of these changes in voltage gating were further quantified. For wild-type 3 homotypic channels, the initial 0.5 seconds of current decay after imposition of a transjunctional voltage of +80 mV was well fit by a single exponential function with a time constant (
) of 240 milliseconds. For 8-G22R and 3 heteromeric channels, the current decay at +80 mV was significantly (P<0.05) faster, with = 95 milliseconds (Fig. 7B). Steady-state junctional conductance was normalized to the values obtained at ±20 mV and fit to the Boltzmann equation. These data showed that the steady-state reductions in conductance for co-injected heteromeric channels were greater than the reductions for homotypic 3 channels (Fig. 7C). Heteromeric 8-G22R and 3 channels exhibited a 35-40% decrease in both the values of V0 and Gjmin compared with 3 homotypic channels (Table 2). These results clearly show that co-expression of 8-G22R with wild-type 3 results in significantly altered gating properties. Since 8-G22R is a functionally silent allele (Fig. 6B), these shifted gating properties are most consistent with heteromeric channels being formed by the co-injected 8-G22R and 3 subunits.
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Discussion |
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8-G22R mutant subunits interact with wild-type 3 subunits to modulate gap junction formation and channel properties that influence the lens homeostasis. We believe that this mechanism also applies to additional connexin point mutations expressed in different organs or cell types, and this work expands a previous hypothesis, suggesting that cellular defects are caused by aberrant trafficking and intracellular accumulation of toxic aggregates composed of mutant connexin proteins (Berthoud et al., 2003). We have shown that the knocked-in 3 subunits prevent severe cataracts and lens posterior rupture by restoring the gap junction structures containing 8-G22R subunits in vivo. Moreover, mutant 8-G22R subunits contributed to functional heteromeric channels with wild-type 3 subunits in paired Xenopus oocytes in vitro. These heteromeric channels showed a reduction in junctional conductance but faster gating kinetics and increased voltage sensitivity compared to homomeric channels formed by wild-type 3 subunits alone. Taken together, these findings suggest that the formation of heteromeric gap junctions by knocked-in 3 and mutant 8-G22R subunits is part of the mechanism that contributes to the suppression of the abnormal lens phenotypes.
It is particularly intriguing that the knocked-in 3 subunits behave differently from the endogenous 3 subunits in their interactions with the 8-G22R mutant. We have previously shown that in 8(G22R/G22R) homozygous mice, endogenous 3 subunits positively contributed to the severity of cataract formation and posterior rupture (Chang et al., 2002). By contrast, the knocked-in 3 subunits actively suppressed cataract formation and posterior rupture. Several differences between knocked-in 3 and endogenous 3 can be derived from previous studies: (1) knocked-in 3 subunits, on the endogenous 8 loci, are expressed earlier than the endogenous 3 subunits; (2) the protein level of knocked-in 3 is higher than that of endogenous 3; (3) in the absence of endogenous 8 connexin, knocked-in 3, but not endogenous 3, forms pH-sensitive channels between the lens fiber cells (Martinez-Wittinghan et al., 2003; Martinez-Wittinghan et al., 2004). This work provides yet another difference: only knocked-in 3 subunits functionally interact with 8-G22R to suppress lens phenotypes.
Both endogenous 3 and knocked-in 3 connexins are phosphorylated in the lens. However, mutant 8-G22R significantly reduced the phosphorylated forms of endogenous 3 connexin (Chang et al., 2002), but did not alter the migration of knocked-in 3 (Fig. 4). Thus, phosphorylation of earlier expressed 3 in the KI3 mice may be one of the crucial factors why only the knocked-in 3 interacts with mutant 8-G22R to target it to gap junctions in vivo. Alternatively, the higher expression level of knocked-in 3 could be another important factor that contributes to the phenotypic rescues in the 3(-/-) 8(G22R/KI3) lenses, because gap junction channels formed by 3 connexin alone are sufficient to maintain the lens transparency when mutant 8 subunits are absent in the lens fibers.
This work provides additional direct evidence supporting the hypothesis that the N-terminal domain of a connexin can modulate both conductance and voltage gating of gap junction channels formed by different subunits. The residue Gly 22 is an evolutionarily conserved residue in the N-terminal domain of 8 connexin. The N-terminal domain has been reported to form a part of the transjunctional voltage sensor and plays a role in gating of gap junction channels (Verselis et al., 1994). Electrophysiology studies of paired Xenopus oocytes provide crucial data showing that mutant 8-G22R subunits can form heteromeric gap junction channels with wild-type 3 subunits. However, these heteromeric channels have reduced macroscopic conductance and faster gating. Thus, these observations might explain why the lens phenotypes are not fully rescued in 3(-/-) 8(G22R/KI3) lenses, which have mild nuclear cataracts.
Various types of cataracts have been linked to different mutations of 3 and 8 connexins in humans and mice. To date, 8 mutations have been reported in the N-terminal domain (G22R and R23T) (Chang et al., 2002; Willoughby et al., 2003), the E1 loop (D47A, E48K, V64G and V64A) (Steele et al., 1998; Berry et al., 1999; Zheng et al., 2005; Graw et al., 2001), the second transmembrane domain (P88S) (Shiels et al., 1998), and the C-terminal intracellular domain (I247M) (Polyakov et al., 2001). Mutations of 3 have been identified in the first transmembrane domain (F32L) (Jiang et al., 2003), E1 loop (P59L and N63S) (Bennett et al., 2004; Mackay et al., 1999), E2 loop (P187L and N188T) (Rees et al., 2000; Li et al., 2004) and C-terminal domain (S380fs) (Mackay et al., 1999). The unique properties of the heteromeric channels formed by wild-type and mutant subunits of 3 or 8 connexins suggest some molecular explanations as to why a variety of cataract types have been reported to be caused by different mutations of 3 or 8 in humans and mice. These data indicate that mutated connexin subunits can act as specific molecular tools to change the properties of gap junction channels and manipulate the cell-cell-communication signals in the lens. Thus, we believe that the mouse mutant lines with different point mutations in 3 or 8 connexins provide extremely useful models for investigating the specific communication signals mediated by gap junction channels in the lens fiber cells and for understanding how changes in fiber-fiber communication lead to distinct cataract phenotypes. Finally, a better understanding of the underlying molecular mechanism for cataract formation will probably lead us to the development of alternative approaches to prevent or delay cataract formation.
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Materials and Methods |
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3(-/-) 8(G22R/-), containing only one mutant 8 G22R allele, and 3(-/-) 8(KI3/-), containing only one 3 knock-in allele, were generated from several generations of breeding the original mutant lines [homozygous Lop10 mutant mice (8-G22R) and 3 knock-in (KI3) mice, respectively] with double-knockout 8(-/-) 3(-/-) mice. Intercross of these two-compound mutant mice generated the following three types of littermates: 3(-/-) 8(G22R/-), 3(-/-) 8(KI3/-) and 3(-/-) 8(G22R/KI3). The genotype of the mutant mice was assessed by PCR as described in previous papers (Gong et al., 1997; Rong et al., 2002; White, 2002).
Lens phenotype and morphological analysis
Mice were euthanized by CO2 asphyxiation according to the ACUC-approved animal protocol. Freshly dissected lenses were imaged under a Leica MZ16 dissecting scope using a digital camera. For morphological analysis by light and electron microscopy, enucleated mouse eyeballs or lenses were fixed in a solution containing 2% glutaraldehyde and 2.5% formaldehyde in 0.1 M cacodylate buffer (pH 7.2) for 5 days at room temperature, followed by postfixation in 1% aqueous OsO4, stained en-bloc with 2% aqueous uranyl acetate, and then dehydrated through graded acetone. Samples were embedded in Epon 12-Araldyte 502 resin (Ted Pella, Redding, CA). Lens-thick sections (1 µm) were collected on glass slides and stained with Toluidine Blue, and histological images were acquired with a Zeiss Axiovert 200 light microscope with a digital camera. Transmission electron microscopy (TEM) data were collected from thin sections (80 nm) stained with 5% uranyl acetate followed by Reynold's lead citrate using a JEOL JEM-1200EX electron microscope (JEOL, Tokyo, Japan).
Immunohistochemical analysis
Mouse eyes were fixed with 4% paraformaldehyde in PBS for 30 minutes on ice, washed with PBS and incubated in 30% sucrose in PBS overnight at 4°C. The samples were embedded in OCT and 8-µm frozen sections were collected for coimmunostaining of 8 connexin and phalloidin. A previously described protocol was used to examine the subcellular localization of 8 connexin (Rong et al., 2002). Images were collected with a laser scanning confocal microscope (Leica).
Biochemical analysis of lens protein
Water-soluble and -insoluble lens proteins were prepared as previously described (Gong et al., 1997). Mouse lenses were homogenized in a solution of 20 mM NaOH and 1 mM Na2CO3 and centrifuged (10,000 g for 15 minutes). The insoluble pellets were washed (first with the same solution followed by a second wash with 1 mM Na2CO3) and dissolved in an equal volume of sample buffer (60 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.001% Bromphenol Blue, 10 mM DTT). Equal volumes of samples were loaded on a 12.5% SDS-PAGE gel for separation, and separated proteins were transferred to a PVDF membrane (Bio-Rad). Expression of 3 or 8 connexins was detected with specific antibodies.
Expression of 3 and 8-G22R in Xenopus oocytes
Wild-type murine 3 connexin was inserted between the XhoI-SpeI sites of the pCS2+ expression vector to conduct functional expression studies in Xenopus laevis oocytes. Mouse 8-G22R was subcloned into the pCS2+ vector by using EcoRI restriction sites. Constructs were linearized with NotI and transcribed by using the SP6 Message Machine kit (Ambion) to obtain cRNAs. Adult Xenopus females were anesthetized with MS222 and ovaries were removed. Stage V-VI oocytes were collected after ovarian lobes were defolliculated in a solution containing 50 mg/ml collagenase B and 50 mg/ml hyaluronidase in modified Barths medium (MB) without Ca2+. Cells were subsequently cultured in MB medium at room temperature. For physiological analysis, cells were first injected with 10 ng of an antisense Xenopus 2 oligonucleotide to eliminate conductance produced by endogenous intercellular channels. After a 24-hour recovery period, antisense oligonucleotide-injected oocytes were re-injected with 3 and 8-G22R cRNAs (5 ng/cell) or in combination or with H2O as a negative control. The vitelline envelopes were then removed in a hypertonic solution (200 mM aspartic acid, 10 mM HEPES, 1 mM MgCl2, 10 mM EGTA, 20 mM KCl at pH 7.4), and the oocytes were manually paired with the vegetal poles apposed in MB medium containing Petri dishes. All cells were bathed in a solution of elevated Ca2+ (2 mM CaCl2 in MB).
Biochemical analysis of oocyte proteins
Oocytes were collected in 1 ml of lysis buffer containing 5 mM Tris pH 8.0, 5 mM EDTA and protease inhibitors. Oocytes were lysed by mechanical passage through a series of needles of diminishing caliber (20, 22 and 26 Ga) using a 1 ml syringe. After homogenization, extracts were centrifuged at 1000 g at 4°C for 5 minutes in a microcentrifuge. The supernatant was transferred to a new tube and centrifuged at 100,000 g at 4°C for 30 minutes in a TLC45 ultracentrifuge. Membrane pellets were resuspended in SDS sample buffer (2 µl per oocyte) and samples were separated on 10% SDS gels and then transferred to nitrocellulose membranes. Blots were blocked with 3% BSA in 1x PBS with 0.02% NaN3 for 1 hour and then probed with a rabbit anti-8 connexin antibody, or a rabbit anti-3 connexin antibody. Alkaline-phosphatase-conjugated goat anti-rabbit IgG (Jackson laboratories) was used as a secondary antibody. Samples were detected using Sigma FastTM alkaline phosphatase substrate (Sigma). Band-intensity values from three independent experiments were normalized to the mean value of intensity for the 8-G22R-injected or 3-injected samples.
Electrophysiology study of paired Xenopus oocytes
Following overnight incubation, gap junctional coupling between oocyte pairs was measured by dual whole-cell voltage clamp. Current- and voltage-electrodes (1.2 mm diameter, omega dot; Glass Company of America, Millville, N.J., USA) were pulled to a resistance of 1-2 m
with a horizontal puller (Narishige, Tokyo, Japan) and filled with solution containing 3 M KCl, 10 mM EGTA and 10 mM HEPES, pH 7.4. Voltage clamping of oocyte pairs was performed with two GeneClamp 500 amplifiers (Axon Instruments, Foster City, Calif., USA) controlled by a PC-compatible computer through a Digidata 1320A interface (Axon Instruments). The pCLAMP 8.0 software (Axon Instruments) was used to program stimulus and data collection paradigms. For measurements of junctional conductance, both cells of a pair were initially clamped at -40 mV to ensure zero transjunctional potential and alternating pulses of ±10-20 mV were imposed to one cell. Current delivered to the cell clamped at -40 mV during the voltage pulse was equal in magnitude to the junctional current, and was divided by the voltage to yield the conductance, Gj=Ij/(V1-V2). To determine voltage-gating properties, transjunctional potentials (Vj) of opposite polarity were generated by hyperpolarizing or depolarizing one cell in 20 mV steps (over a range of ±120 mV), while clamping the second cell at -40 mV. Currents were measured at the end of the voltage pulse, at which time they approached steady state (Ijss), and the macroscopic conductance (Gjss) was calculated by dividing Ijss by Vj. Gjss was then normalized to the values determined at ±20 mV, plotted against Vj and fit to a Boltzmann equation of the form: Gjss=(Gjmax-Gjmin)/1+exp[A(Vj-V0)]+Gjmin, where Gjss is the steady-state junctional conductance, Gjmax (normalized to unity) is the maximum conductance, Gjmin is the residual conductance at large values of Vj, and V0 is the transjunctional voltage at which Gjss=(Gjmax-Gjmin)/2. The constant A (=nq/kT) represents the voltage sensitivity in terms of gating charge as the equivalent number (n) of electron charges (q) moving through the membrane, k is the Boltzmann constant, and T is the absolute temperature.
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Acknowledgments |
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