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Department of Physiology and Biophysics, Rosalind Franklin School of Medicine and Science, North Chicago, Illinois
Correspondence: Address reprint requests to Lisa Ebihara, Finch University of Health Sciences, The Chicago Medical School, Dept. of Physiology and Biophysics, 333 Green Bay Rd., North Chicago, IL 60064. Tel.: 847-578-3424; E-mail: lisa.ebihara{at}rosalindfranklin.edu.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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).
Three different connexins have been identified in the mammalian lens: Cx43, Cx46, and Cx50 (Beyer et al., 1987; Paul et al., 1991; White et al., 1992). Cx43 is expressed primarily in the epithelial cells, whereas Cx46 and Cx50 are coexpressed in fiber cells. All three of these gap junctional proteins are believed to play an important role in maintaining metabolic homeostasis in the lens. It has recently been shown that mutations in the genes for Cx50 and Cx46 are associated with congenital cataracts in humans (Shiels et al., 1998; Mackay et al., 1999). Furthermore, studies on genetically manipulated mice demonstrate that disruption of the gene for either Cx50 or Cx46 by homozygous knockout leads to the development of cataracts in mice (Gong et al., 1997; White et al., 1998). However, the mice had different phenotypes. Cx50 knockout mice exhibited mild nuclear cataracts and microopthalmia. In contrast, Cx46 knockout mice developed severe nuclear cataracts but lens and eye growth were normal. Replacement of Cx50 with Cx46 by knockin rescued lens clarity but could not restore normal growth, suggesting that the two connexins may be tailored to serve specific functions in the lens (White, 2002).
The functional properties of gap junctional channels formed by Cx50 and Cx46 have been examined in communication-deficient mammalian cell lines and Xenopus oocyte pairs using electrophysiological techniques (White et al., 1994; Paul et al., 1991; Srinivas et al., 1999; Hopperstad et al., 2000; Xu and Ebihara, 1999; Eckert, 2002; Sakai et al., 2003). These studies show that although Cx50 and Cx46 have very similar amino acid sequences, their gap junctional channels exhibit marked differences in voltage-gating properties and single-channel conductance. Furthermore, single oocytes injected with cRNA for Cx50 or Cx46 develop calcium-sensitive hemichannel currents with distinct voltage-gating properties (Ebihara and Steiner, 1993; Beahm and Hall, 2002).
The N-terminus may play an important role in voltage-dependent gating of gap junctional channels. Previous mutational studies of Cx32 and Cx26 have shown that charged amino acids in the N-terminus determine the polarity of Vj-gating (Verselis et al., 1994; Oh et al., 2000; Purnick et al., 2001). More recently, Musa et al. (2004) reported that two charged amino acids in the N-terminus of Cx40 and Cx43 control the Vj-gating polarity and block by spermine of these connexins. In this study, we constructed a series of chimeras composed of segments of rat Cx46 and chicken Cx45.6, which is a species ortholog of mouse Cx50 (Jiang et al., 1994), expressed them in Xenopus oocyte pairs or N2A cells, and examined the resulting gap junctional conductances. Our results demonstrate that the N-terminus is largely responsible for the change in voltage-gating properties. In addition, exchanging the N-terminus modifies single gap junctional channel conductance.
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are delimited as follows. Cx46 is divided into N (Met1–Lys23), M1 (Val24–Ala41), E1 (Glu42–Arg76), M2 (Phe77–Gly94), L (His95–Ala150), M3 (Leu151–Phe169), E2 (Ile170–Thr207), M4 (Ile208–Leu226), and CT (Glu227–Ile416). Cx45.6 is divided into N (Met1–Arg23), M1 (Val24–Ala41), E1 (Glu42–Arg76), M2 (Leu77–Gly94), L (His95–Thr148), M3 (Leu149–Phe167), E2 (Ile168–Thr205), M4 (Ile206–Val224), and CT (Glu225–Val400). To generate the Cx45.6-46NT construct, two primers were synthesized to amplify the DNA encoding the N-terminus (NT) of Cx46: sense 5'-CAATTAGGCTTGTACATATTGTCGTTAGAACGC-3'; and antisense, 5'-CAGCACCAGAATTCGAAAGATGAACAGGACGGTC-3'. The sense primer corresponded to a region of the transcription vector, SP64T, containing a naturally occurring BsrGI restriction site. The antisense primer corresponded to codons 27–37 in M1 of Cx46. The nucleotide sequence of codons 32–34 in the antisense primer were modified to create a BstBI restriction site without changing the predicted amino acid sequence (PheArgIle). A second set of primers was designed to amplify Cx45.6 sequences, as well as the transcription vector, SP64T, in which Cx45.6 was subcloned: sense, 5'-CTCTTCATTTTTCGAATCCTGATCCTGGGAACGGCTG-3'; and antisense, 5'CGACAATATGTACAAGCCTAATTGTGTAGCATCTG-3'. The sense primer corresponded to the last 12 codons in M1 of Cx45.6. The codons for the conserved PheArgIle sequence in Cx45.6's M1 were also modified to generate a BstBI restriction site without changing the predicted amino acid sequence. DNA amplification was performed with PfuTurbo DNA Polymerase (Stratagene, La Jolla, CA) according to the manufacturer's protocol in a thermocycler (PTC-150, MJ Research, Watertown, MA). The PCR products were digested with BsrGI and BstBI, gel purified, and ligated together to generate a chimera in which the N-terminus of Cx45.6 (amino acids 1–23) was replaced with the corresponding NT of Cx46. A similar strategy was used to generate the other chimeras. Point mutations were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's protocol. All of the constructs were sequenced (DNA Sequencing and Synthesis Facility, Iowa State University, Ames, IA) to ensure that PCR amplification did not introduce random mutations.
Expression of connexins in Xenopus oocytes
Adult female Xenopus laevis frogs were anesthetized on ice and a partial ovarectomy performed. The oocytes were defolliculated by treating them with collagenase IA (Sigma Chemical, St. Louis, MO). Stage V and VI oocytes were selected and pressure injected using a Nanoject variable microinjection apparatus (model 3-000-203, Drummond Scientific, Broomal, PA) with 0.23 ng of oligonucleotides antisense to mRNA for Xenopus Cx38 as previously described (Ebihara, 1996). The oocytes were incubated overnight at 18°C in Modified Barth's Solution containing (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 15 Hepes, .3 CaNO3 x 4H2O, 0.41 CaCl2 x 6H2O, 0.82 MgSO4 x H2O, 550 mg/L pyruvate and 50 µg/ml gentamycin, pH 7.4. Connexin cRNAs were synthesized using the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX) according to the manufacturer's instructions. The amount of cRNA was quantitated by measuring the absorbance at 260 nm. The purity and amount of cRNA was further assessed by agarose gel electrophoresis. Cx38 antisense-pretreated oocytes were injected with 36 nl of 0.04–4 ng/nl connexin cRNA and incubated for an additional 16–24 h at 18°C in Modified Barth's Solution containing 5 mM CaCl2.
Expression of connexins in N2A cells
N2A mouse neuroblastoma cells were grown in Dulbecco's minimal essential medium (Life Technologies, Gaithersburg, MD) containing 10% fetal bovine serum, 2 mM L-Glutamine, 100 units/ml penicillin G and 100 µg/ml streptomycin sulfate in a humidified 5% CO2 incubator at 37°C.
For transient transfections, N2A cells (grown to 
60% confluence on lysozyme-treated coverslips in 35-mm tissue dishes) were cotransfected with 1 µg of Cx45.6 or Cx45.6-46NT cDNA and 1 µg of enhanced green fluorescence protein (EGFP) cDNA using Superfect reagent (Qiagen, Valencia, CA) following the manufacturer's protocol. Electrophysiological recordings were performed 18–72 h later. Cell pairs expressing exogenous connexins were identified by EGFP fluorescence using a Nikon Diaphot microscope (equipped with a mercury arc lamp and fluorescein isothiocyanate filter sets).
Electrophysiological measurement and analysis of gap junctional currents expressed in Xenopus oocytes
For gap junctional conductance measurement, connexin cRNA-injected oocytes were manually devitellinized and paired as previously described (Ebihara, 1992). The oocyte pairs were allowed to incubate at room temperature for 2–4 h (or in some cases overnight) before electrophysiological recording. Double two-microelectrode voltage-clamp experiments were performed using a Gene-Clamp 500 and an Axoclamp 2A voltage-clamp amplifier (Axon Instruments, Union City, CA). The microelectrodes were filled with 3 M KCl and had resistances between 0.1 and 0.6 M
. To prevent electrode leakage, the tips of the electrodes were backfilled with 1% agar in 3 M KCl. For simple measurements of gap junctional coupling, both cells of the pair were initially held at –40 mV and 5- to 10-mV steps were applied to one cell while holding the second cell at –40 mV. Under these conditions, the change in current measured in the second cell would be equal to the junctional current (Ij). The junctional conductance (Gj) was calculated as (Gj = Ij/Vj), where Vj = Vcell 2 – Vcell 1. To determine the voltage-dependent gating properties of the gap junctions, families of junctional current traces were recorded in oocyte pairs by applying transjunctional voltage-clamp steps between +90 mV and –90 mV in decrements of 20 mV. Changes in junctional conductance during the experiment were monitored by applying a 5-mV prepulse of 1-s duration 1 s before the initiation of the test pulse. Only cell pairs with junctional conductances <7 µS were selected for analysis. The instantaneous junctional current (Ij,inst) was determined by fitting the junctional current traces to the sum of two exponentials and extrapolating back to the beginning of the voltage-clamp pulse. The steady-state junctional current (Ij,ss) was determined at the end of the pulse. Instantaneous conductance (Gj,inst) was calculated for each voltage trace as (Gj,inst = Ij,inst/Vj) and normalized to the conductance of the prepulse for each trace. The normalized steady-state junctional conductance (Gj,ss) was calculated as (Gj,ss = Ij,ss/Ij,inst). The relationship between Gj,ss and Vj was fit using a two-state Boltzmann equation (Spray et al., 1981):
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k is the Boltzmann constant, T = absolute temperature, and q = elementary charge. Pulse generation and data acquisition were performed using a PC computer equipped with PCLAMP 6 software and a TL-1 acquisition system (Axon Instruments). Currents were filtered at 20–50 Hz and digitized using PCLAMP6 software and a Digidata 1200 interface (Axon Instruments). All experiments were performed at room temperature (20–22°C).
Electrophysiological measurement and analysis of gap junctional channels expressed in N2A cells
Junctional currents were recorded from transiently transfected N2A cell pairs using the dual whole-cell patch-clamp technique (Neyton and Trautmann, 1985) and two patch-clamp amplifiers (Axopatch 200A, Axon Instruments; and EPC-7, List Electronic, Darmstadt, Germany). Patch pipettes were pulled from glass capillaries on a Flaming/Brown Micropipette puller (model P-87; Sutter Instruments, Novato, CA). The patch electrodes had resistances of 3–8 M when filled with standard internal solution containing (in mM) 130 CsCl, 10 EGTA, 0.5 CaCl2, 3 MgATP, 2 Na2ATP, 10 HEPES, pH 7.5. The extracellular solution contained (in mM) 140 NaCl, 2 CsCl, 2 CaCl2, 1 MgCl2, 4 KCl, 5 dextrose, 2 pyruvate, 1 BaCl2, 5 HEPES, pH 7.5, To measure gap junctional conductance, both cells of a pair were initially held at a common holding potential of 0 mV. Voltage pulses of 4- to 8-s duration between 100 mV and –100 mV were applied to one cell whereas the second cell was held at 0 mV. Gap junctional current was recorded in the second cell of the pair. The normalized steady-state junctional conductance (Gj,ss) was determined by dividing the steady-state current (measured at 8 s) by the initial current. No series compensation was used. Only cell pairs having conductances <7 nS were selected for data analysis to minimize series resistance artifacts.
Single-channel data were obtained from cell pairs that had only one or two functional channels using voltage-clamp protocols as described above. All-amplitude histograms were constructed from the patch-clamp records and fit to the sum of Gaussians to determine the amplitude of the current during channel openings. Data was acquired using a PC computer equipped with a Digidata 1320A interface and PCLAMP8 or 9 software (Axon Instruments). The current signal was filtered at 1 kHz and digitized at 5 kHz.
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RESULTS |
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1 = 80.7 ± 10.3 ms and 2 = 1926.7 ± 123.9 ms at Vj = –90 mV. In contrast, Cx46 junctional currents inactivated slowly (1 = 437.7 ± 58.6 ms and 2 = 2023.6 ± 46.7 ms at Vj = –90 mV). The two types of channels also showed differences in steady-state conductance-voltage relationships, which were quantified by fitting the data to a Boltzmann equation (Table 2). The conductance-voltage relationship of Cx45.6 showed a more pronounced voltage sensitivity (half-inactivation potential, V0 = 35.4 mV, slope factor, A = .149) than Cx46 junctions (V0 = 59.5 mV, A = .084).
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; Oh et al., 2000). Thus, changing amino acids in this region might be expected to influence single gap junctional channel conductance as well as channel gating parameters. To test this hypothesis, Cx45.6-46NT and wild-type Cx45.6 were expressed in communication-deficient N2A cells. Dual whole-cell patch-clamp experiments showed that both constructs formed functional gap junctional channels. Fig. 6 compares representative families of macroscopic current traces, I-V curves, and normalized steady-state Gj-Vj curves recorded from cell pairs expressing wild-type Cx45.6 or Cx45.6-46NT. The behavior of Cx45.6 and Cx45.6-46NT in N2A cells was similar to that observed in oocytes. One of the main effects of exchanging the N-terminus was to slow the inactivation kinetics. The time course of inactivation of Cx45.6 and 46NT at Vj = 100 mV could be described by a single exponential with = 117.9 ± 12.1 ms and 907.5 ± 129 ms, respectively. It also reduced the voltage sensitivity of the steady-state conductance (Table 2).
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j, of 184 ps. Because of limitations in time resolution, it was not possible to accurately measure the amplitude of the main state for Vj 
|100 mV|. In four cell pairs containing one or two functional channels, j,main = 202.4 ± 9.7 pS.
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j,main and altered channel gating kinetics. Fig. 8 A shows representative junctional current traces recorded from a Cx45.6-46NT cell pair at Vj = 100, 90, 70, 60, and 50 mV. The junctional current exhibited three or four discrete levels corresponding to the main state (solid line), and substate 1, substate 2, and closed state (dashed lines). Fig. 8 C shows the I-V relationships for the main state and the subconductance states. In this particular experiment, j,main = 116.4 pS, j,s1 = 35.2 pS, and j,s2 = 17.8 pS. Analysis of data from eight cell pairs containing one or two functional channels yielded a j,main = 129.6 ± 4.0 pS, which is significantly reduced compared to that observed for the Cx45.6 channels. Another difference was that the open times for the main conductive state were prolonged compared to those of Cx45.6 (Fig. 9). Recordings at Vj = ±100 mV yielded a mean open time of 1240.6 ms. In contrast, the mean open time of Cx45.6 channels was 64 ms.
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DISCUSSION |
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; Xu and Ebihara, 1999). Exchanging the N-terminus of Cx45.6 with that of Cx46 reduces the main-state conductance to 130 pS, which is similar to the value previously reported for Cx46 (Hopperstad et al., 2000). We attempted to identify discrete determinants within the N-terminus by constructing a series of chimeras in which we introduced short segments of Cx46 sequence into the Cx45.6 template. Our results identified a uniquely occurring arginine at position 9 in Cx46 as the main determinant in the N-terminus. Introducing just this one amino acid into the N-terminus of Cx45.6 could account for most of the changes in voltage-gating properties of the gap junctional channels. R9 is conserved in all of the Cx46 species orthologs and is absent in Cx45.6 and other species orthologs of Cx50, supporting its key role in determining the voltage-dependent gating properties and single-channel conductance of the lens fiber connexins.
It should be mentioned that although the N-terminus could account for most of the functional differences observed between Cx45.6 and Cx46, it could not account for all of them. For example, the steady-state junctional conductance of the Cx45.6-46NT had a voltage sensitivity that was intermediate between that of Cx45.6 and Cx46. This suggests that there are other regions of the connexin besides the N-terminus that contribute to voltage gating.
To better understand how the Vj -gating properties of the gap junctional channels are derived from the gating properties of their component hemichannels, we compared the macroscopic voltage-gating properties of heterotypic Cx45.6/46NT gap junctional channels with those of the corresponding homotypic junctional channels. Junctional currents from Cx45.6/46NT cell pairs showed asymmetric voltage-dependent closure, characterized by a faster time course when Vj was positive with respect to the Cx45.6 oocyte. The steady-state Gj-Vj relationship was also asymmetric. The junctional conductance declined more steeply at positive transjunctional voltages relative to the Cx45.6 oocyte. Similar results were obtained for heterotypic Cx45.6/N9R junctional cell pairs (data not shown). This behavior could be predicted from that of the homotypic channels if it is assumed that in a gap junction, the component hemichannels function independently and that both hemichannels close in response to positive potential.
It has been previously proposed that a portion of the N-terminus lies within the pore of the channel when the channel is open and can sense membrane field, and that movement of the N-terminus toward the cytoplasm causes the channel to close (Verselis et al., 1994; Oh et al., 2000). According to this model, replacing a neutral residue with a positively charged one would make the N-terminus of the Cx45.6 subunit less negative and thus would be predicted to reduce the Vj sensitivity of Cx45.6 in agreement with our experimental data. The addition of a positive charge near the cytoplasmic end of the Cx45.6 hemichannel pore would also depress the entry of cations into the channel and could account for the experimentally observed reduction in the unitary conductance of the main open state in homotypic 46NT/46NT cell pairs. An important question that remains to be addressed is whether exchanging the N-terminus alters the channel's permeability to biological relevant signaling molecules. It would also be interesting to determine if genetic replacement of the coding region of the rodent version of Cx45.6 with a chimera containing the N-terminus of Cx46 modifies ocular growth or leads to the development of cataracts in mice.
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ACKNOWLEDGEMENTS |
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Submitted on January 7, 2004; accepted for publication July 7, 2004.
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Paul, D. L., L. Ebihara, L. J. Takemoto, K. I. Swenson, and D. A. Goodenough. 1991. Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J. Cell Biol. 115:1077–1089.
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