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CELLULAR |
Departments of
1 Physiology & Biophysics
2 Cell Biology & Anatomy, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA
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Abstract |
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(Received 18 May 2006;
accepted after revision 23 August 2006;
first published online 24 August 2006)
Corresponding author L. Ebihara, Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA. Email: lisa.ebihara{at}rosalindfranklin.edu
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Introduction |
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Gap junctional channels formed from different connexins have different biophysical properties, such as unitary conductance, voltage gating and molecular permeability. Previous studies have shown that gap junctions are permeable to a number of important biological signalling molecules such as cAMP, IP3 and glutamate (Lawrence et al. 1978; Saez et al. 1989). Different connexin channels show significant differences in permeability to metabolites and second messengers (Goldberg et al. 1999, 2002; Niessen et al. 2000; Bevans et al. 1998; Bedner et al. 2006). For instance, by using cyclic nucleotide-gated channels to quantify gap junction-mediated diffusion of cyclic AMP, Bedner et al. (2006) showed that Cx43 gap junctional channels were about 5–6 times more permeable to cAMP than Cx32 or Cx45 channels, and more than 30 times more permeable than Cx36 channels.
To better understand the contribution of charge and steric hindrance to connexin selectivity, fluorescent tracers of different charge and size have been used (Elfgang et al. 1995; Veenstra et al. 1995; Cao et al. 1998; Trexler et al. 2000; Weber et al. 2004). These studies show that certain connexins such as Cx45 and Cx46 form gap junctional channels that are more permeable to the cationic dyes than to the anionic dyes, while other connexins such as Cx43 show little charge preference. Recently, Weber et al. (2004) quantitatively examined the size selectivity of homotypic and heterotypic gap junctional channels expressed in Xenopus oocyte pairs by using a series of Alexa probes that have a similar structure and charge, but differ in size. The results of this study suggest that the size cut-off limits of the connexin channels could be ranked as: Cx32 
Cx43 >> Cx26 Cx40 
Cx45 Cx37 (Weber et al. 2004).
Each connexin has four hydrophobic, transmembrane regions (M1–M4). The N- and C-termini as well as the central loop that connects M2 and M3 are located on the cytoplasmic side of the membrane (Zimmer et al. 1987; Goodenough et al. 1988; Hertzberg et al. 1988; Milks et al. 1988; Yancey et al. 1989). Mutagenesis studies suggest that the N-terminus plays a critical role in determining the biophysical properties of gap junctional channels. The amino terminus has been shown to act as the voltage sensor and determine the polarity of transjunctional voltage (Vj)-dependent gating of Cx32 and Cx26 (Verselis et al. 1994; Oh et al. 1999, 2000; Purnick et al. 2000a, 2000b). It also is an important determinant of unitary conductance and rectification (Oh et al. 1999). The first 10 amino acid residues of the N-terminus of Cx32 have been proposed to lie within the channel pore and form the channel vestibule (Purnick et al. 2000a,b). Several recent studies suggest that the N-terminus may play a similar role for the 
-group connexins (Musa et al. 2004; Tong et al. 2004; Srinivas et al. 2005).
In the present study, we examine the structural basis for the biophysical properties of two lens gap junctional proteins, chicken Cx45.6 (which is the chicken orthologue of Cx50) and rat Cx43, by constructing a chimera in which the N-terminus of Cx45.6 was exchanged with the corresponding domain of Cx43. Our results suggest that the N-terminus plays a critical role in determining many of biophysical properties of Cx45.6 gap junctional channels, including molecular selectivity for larger permeants.
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Methods |
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We constructed a Cx45.6–43NT chimera in which the N-terminus of chicken Cx45.6, delimited as Met1–Arg23 (Bennett et al. 1991), was replaced by the corresponding domain of rat Cx43. Two steps were used to generate the Cx45.6–Cx43NT chimera. (1) The region (Met1–Thr39) corresponding to the N-terminus and a portion of M1 of Cx45.6 was replaced with the corresponding region of Cx43. This exchange was facilitated by the presence of a naturally occurring PvuII restriction site at the same position in the M1 domain of both Cx45.6 and Cx43 and a second naturally occurring PvuII restriction site in the transcription vector, SP64T. (2) There are two amino acid differences in the M1 domain between the chimera constructed in the first step and Cx45.6. These two amino acids (Ser, Leu) of the chimera were replaced with the corresponding amino acids (Thr, Ile) of Cx45.6 by PCR mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the primers: sense 5'-GGGAAGGTGTGGCTGACAGTGCTCTTCATATTC-AGAATCCTGATCCTGGGGACAGCTG-3'; and antisense 5'-CAGCTGTCCCCAGGATCAGGATTCTGAATATGAA-GAGCACTGTCAGCCACACCTTCCC-3'. The construct was fully sequenced (DNA Sequencing and Synthesis Facility, Iowa State University, Ames, IA, USA) to ensure that PCR amplification did not introduce random mutations.
Transient transfection of connexins in N2A cells
N2A mouse neuroblastoma cells were grown in Dulbecco's modified Eagle medium containing high glucose with 2 mM L-glutamine and no sodium pyruvate (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, 100 u ml–1 penicllin G, and 100 µg ml–1 streptomycin sulphate, in a humidified atmosphere of 5% CO2 at 37°C. For transient transfection, 1 µg of chicken Cx45.6, rat Cx43 or Cx45.6–Cx43N was cotransfected with 1 µg of enhanced green fluorescence protein (EGFP) cDNA into N2A mouse neuroblastoma cells using Superfect Transfection Reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Patch-clamp experiments were conducted 18 h later on EGFP-positive cell pairs.
Stable transfection of connexins in HeLa cells
A clone of HeLa cells essentially devoid of connexins was provided by V.K. Verselis (Albert Einstein College of Medicine, Bronx, NY, USA). Stable HeLa cell lines expressing chicken Cx45.6, rat Cx43 or Cx45.6–43N were generated using the Flp-In System (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. The stably transfected cells were selected and maintained in cell culture medium containing 300 µg ml–1 hygromycin.
Immunofluorescence localization
HeLa cells stably expressing Cx45.6, Cx43 and Cx45.6–Cx43N were seeded on glass coverslips (gelatinized, carbon-coated) at 20% confluence. At 2 days post-plating, cells were washed with phosphate-buffered saline (PBS), fixed in methanol at –20°C for 10 min, and stored in methanol at –4°C prior to immunofluorescent staining.
The polyclonal rabbit primary antibodies include anti-Cx45.6 antibody (provided by Dr Jean X. Jiang (University of Texas Health Science Center, San Antonio, TX, USA) against the C-terminus of Cx45.6, and anti-Cx43 antibody (Zymed Laboratories South San Francisco CA, USA) against the C-terminus of Cx43. Coverslips with fixed cells were washed three times with PBS for 5 min, followed by incubation with primary antibodies (1: 200 diluted) at 37°C for 2 h. Then the cells were washed with PBS for at least 30 min and incubated in secondary antibody (Texas red-conjugated goat antirabbit IgG, Jackson ImmunoResearch Laboratories; 1: 50 diluted) at 37°C for 1 h. After extensive PBS washes over 30 min, coverslips were mounted onto glass slides. The specimens were examined, and images were recorded through a Leica DMRB microscope equipped with a Hamamatsu CCD camera driven by the Openlab imaging program (Improvision).
Electrophysiological recording and analysis of gap junctional channels expressed in N2A cells
Junctional currents were obtained using the dual whole-cell patch-clamp technique as previously described (Xu et al. 2002). Computer-controlled patch-clamp amplifiers (MultiClamp 700 A, Molecular Devices, Sunnyvale, CA, USA) were used to control membrane potential and measure gap junctional currents. The resistance of patch pipettes was 4–6 M
when filled with standard internal solution. The standard internal solution contained (mM): 130 CsCl, 10 EGTA, 0.5 CaCl2, 3 MgATP, 2 Na2ATP, 10 Hepes, pH 7.4. The extracellular solution contained (mM) 140 NaCl, 2 CsCl, 1 MgCl2, 5 Hepes, 4 KCl, 5 dextrose, 2 pyruvate, 1 BaCl2, PH 7.4. Both cells of a EGFP-positive pair were initially held at 0 mV. A series of 8 s voltage-clamp pulses ranging between 100 mV and –100 mV in 10 mV decrements were applied to one cell, while the other cell was held constant at 0 mV and the junctional current was recorded from the non-pulsed cell. The normalized steady state conductance (Gj,ss) was calculated by dividing steady state junctional current (Ij,ss) by initial junctional current (Ij,Institute). The relationship between Gj,ss and Vj was described and fitted by two-state Boltzmann equation:
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| (1) |
Single junctional channel currents were recorded from cell pairs having only one or two active gap junctional channels. The amplitude of single channel current was measured from all-amplitude histogram after fitting the patch clamp records by the sum of Gaussians. Data were obtained and analysed using PCLAMP 9.2 (Molecular Devices) and Sigma Plot 8.0 (SPSS, Chicago, IL, USA) software. All the experiments were conducted at room temperature (20–22°C). Results were presented as mean ± S.E.M.
Dye permeability measurements
Dye permeability through gap junctional channels was studied in stably transfected HeLa cell pairs. The concentration of LY (MW 457, 2 negative charges) dissolved in standard internal solution was 2 mmol l–1. The Alexa dyes (Molecular Probes, Eugene, OR, USA) including Alexa 350 (MW 350, 1 negative charge), Alexa488 (MW 570, 1 negative charge) and Alexa 594 (MW 760, 1 negative charge) were also dissolved in pipette solution to reach a concentration of 1 mmol l–1. The pH of all the dye containing solutions was 7.4. The resistance of patch pipettes ranged between 1 and 1.5 M. Fluorescence was visualized using a Nikon Eclipse TE2000 inverted microscope equipped with an epi-fluorescence attachment (Nikon, Melville, NY, USA). Cells were excited with light from a mercury lamp after passing through appropriate bandpath filters. Image acquisition and analysis were performed using a Spot RT CCD camera (Diagnostic Instruments, Sterling Heights, MI, USA) and v3.5 Spot (Diagnostic Instruments) and Image J software (National Institutes of Health, Bethesda, DC, USA). To determine the relationship between the concentration of fluorescent dye and fluorescence intensity, a calibration curve for the camera was constructed by placing a 10 µl drop of a known concentration of LY on a coverslip and placing a second coverslip over it. Images were taken near the centre of the drop using a fixed exposure time for various concentrations of LY. The images were corrected for background intensity. The resultant calibration curve was linear for LY concentrations less than 2 mM (Fig. 7B).
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To estimate the fluorescent dye permeability through gap junction channels, the dye transfer data were fitted to the two-compartment kinetic model shown in Fig. 1 using Micro Math Scientist 3.0 software (Micromath Research, St Louis, MI, USA).
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| (2) |
KAB is related to the permeability coefficient, P, by
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| (3) |
0 mV. At this potential, there was no substate occupancy and the open probability of the gap junctional channels was close to 1.0 (see supplementary Fig. S1, which is published as supporting information on the J Physiol website). Similar results have been previously reported for hCx50 (Xu et al. 2002), mouse Cx50 (Srinivas et al. 1999) and rat Cx43 (Valiunas et al. 2002). Under these conditions, the number of functional gap junctional channels, N = Gj/
j. Therefore the single channel permeability
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Results |
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To examine the role of the N-terminus in determining the biophysical properties of Cx45.6 and Cx43 gap junctional channels, we constructed a chimeric gap junctional protein (Cx45.6–Cx43N) in which the N-terminus of Cx45.6 was replaced with the corresponding domain of Cx43. The membrane topology of Cx45.6–Cx43N is shown in Fig. 2A. Figure 2B shows a sequence alignment of the N-terminal domain of Cx45.6 and Cx43.
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Figure 4 shows single gap junctional currents recorded at Vj = 90 mV and corresponding unitary I–V curves for Cx45.6, Cx43 and Cx45.6–43 N. Cx45.6–43N channels exhibited prolonged open times compared to Cx43 or Cx45.6. Nearly all of the Cx45.6–43N gating transitions occurred between the main open state and the fully closed state. The unitary Ij–Vj relationship for Cx45.6–43N was linear over the voltage range between –100 mV and +100 mV, and had a slope conductance (j,main) of 101 ± 3.1 pS (n = 6), which was very similar to the value of j for Cx43 (90 ± 0.44 pS, N = 3) but half as large as that for Cx45.6 (j = 202 ± 9.7 pS, N = 4). These data indicate that the N-terminus is also an important determinant of single channel conductance.
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To evaluate whether exchanging the N-terminus of Cx45.6 alters the molecular permeability properties of Cx45.6 gap junctional channels, we initially used the negatively charged dye, Lucifer Yellow (LY). The dye permeability experiments were performed on stably transfected HeLa cell pairs. Expression of the connexins was verified by immunofluorescence analyses with specific antibodies to the corresponding connexins. HeLa cell clones transfected with Cx45.6, Cx43 or Cx45.6–43N displayed immunoreactivity at the appositional plasma membranes as well as intracellularly, confirming that the expressed connexins reached the cell surface and formed gap junctional plaques, as illustrated in Fig. 5.
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40 nS). After 6 min of LY loading, the dye intensity in the recipient cell of both the Cx43 and Cx45.6–43N cell pairs was much stronger than that in the Cx45.6 cell pair. These data suggest that movement of LY through Cx43 and Cx45.6–43N gap junctional channels is faster than that through Cx45.6 channels.
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To examine if swapping the N-terminus altered LY permeability by increasing the size exclusion limit of the Cx45.6–43N channels, we tested the ability of the wild-type and chimeric gap junctional channels to transfer three Alexa dyes (Alexa 350, MW 350; Alexa 488, MW 570; Alexa 594, MW 760) that had similar charge and molecular structure, but ranged in size from MW 350–760. Figure 9A–C shows representative fluorescent images for the time courses of dye transfer for each Alexa dye in cell pairs expressing Cx45.6, Cx43 or Cx45.6–43N. To facilitate comparison of the relative rates of dye transfer, all the images were taken from cell pairs that had similar values of Gj.
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Discussion |
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The mechanisms underlying the differences in molecular permeability properties could involve steric factors. These factors include the length and average diameter of the pore, as well as more localized constrictions. In the present study, we find an inverse relationship between molecular size exclusion limits and the single channel conductance of Cx45.6 and Cx45.6–43N channels, suggesting that the differences in single channel conductance and permeability cannot be accounted for by a change in the average diameter of the pore. Rather, the presence of the N-terminus within the cytoplasmic vestibule of the pore might cause a more localized constriction that could restrict the passage of larger molecules such as Alexa 488 or Alexa 594 but have little effect on conductance. A similar inverse relationship has been seen for other connexin channels, such as Cx26 and Cx32, where Cx26, which has the smaller pore, has the greater unitary conductance (Bevan et al. 1998; Weber et al. 2004).
Electrostatic factors could also cause alterations in single channel conductance and molecular permeability. These factors include interaction of the charged permeant with the wall of the pore, and concentration of cations within the channel vestibule. Comparison of the amino acid sequence of the N-terminus of Cx45.6 with the homologous amino acid sequence of Cx43 reveals 11 amino acid substitutions, with three involving (acidic–basic) charge differences. Exchanging the N-terminus of Cx45.6 for the corresponding domain of Cx43 would make the N-terminus less negative, and thus might reduce accumulation of cations near the cytoplasmic mouth of the channel and result in a decrease in single channel conductance as observed experimentally, provided that the channel exhibits substantial selectivity for cations over anions elsewhere in the permeation pathway. The ionic selectivity of Cx45.6 gap junctional channels has not been investigated. However, gap junctional channels composed of a closely related connexin (Cx46) have been reported to select for K+ over Cl– at a 7: 1 ratio (Trexler et al. 2000). An analogous mechanism has been proposed to account for the large conductance of BK channels (Brelidze et al. 2003).
Previous studies suggest there are other domains of the connexin in addition to the N-terminus that can influence the unitary conductance and molecular selectivity of large molecules. These domains include the carboxyterminal half of the first transmembrane-spanning domain (M1) (Hu & Dahl, 1999; Hu et al. 2006; Ma & Dahl, 2006; Kronengold et al. 2003; Oh et al. 1997), the aminoterminal half of the first extracellular domain (E1) (Kronengold et al. 2003), the carboxyl terminus (CT) (Fishman et al. 1991; Lampe et al. 2000) and possibly the third transmembrane-spanning domain (M3) (Skerrett et al. 2002). The carboxyterminal half of M1 has been recently shown to be a critical determinant of both size exclusion limits and the unitary conductance of connexin channels. Interestingly, chimeras in which the M1 domain of Cx46 was replaced with the corresponding domain of Cx32, or vice versa, showed a correlation between unitary conductance and size exclusion limits as would be predicted to occur in a cylindrical channel with exclusively steric constraints (Ma & Dahl, 2006). Another important domain is the aminoterminal half of E1. Kronengold et al. (2003) showed that fixed charges within E1 influence the single channel conductance, rectification and selectivity of Cx46 hemichannels. This behaviour may be hemichannel specific since these charged residues would be located near the centre of the pore of a gap junctional channel, and therefore could no longer concentrate ions as they do at the extracellular mouth of a hemichannel.
In addition to altering the single channel conductance and molecular permeability, exchanging the N-terminus slowed the inactivation kinetics and reduced the steady-state Vj of the Cx45.6–43N gap junctional channels. It has been proposed that the N-terminus may lie within the pore where it can sense the membrane field and act as a voltage sensor for Vj gating of gap junctional channels (Verselis et al. 1994). The movement of the N-terminus toward the cytoplasm in response to a transjunctional voltage difference is thought to close the channel. In our case, the reduction in transjunctional voltage sensitivity of Cx45.6–43N gap junctional channels could be due to the fact that the N-terminus of Cx43 is more positively charged than that of Cx45.6.
Despite the importance of the N-terminus, it cannot account for all of the differences in voltage-gating properties between Cx45.6 and Cx43, suggesting that there are other critical determinants in Cx43 that influence voltage gating. Comparison of the voltage-gating properties of wild-type Cx43 and Cx45.6–43N gap junctional channels showed several interesting differences: (1) Cx45.6–43N gap junctional channels were less sensitive to junctional voltage, and inactivated more slowly than Cx43 when Vj was high; (2) the open time of single channel current of Cx45.6–43N was longer than Cx43 at high Vj; (3) nearly all of the Cx45.6–43N gating transitions occurred between the main open state and the fully closed state, whereas for Cx43 channels, transitions between the main open state and the residual state predominated. Interestingly, the voltage-gating properties of Cx45.6–43N junctional channels show a strong resemblance to those of the ‘tailless’ mutant rat Cx43 (Revilla et al. 1999; Moreno et al. 2001). The carboxyl terminus of Cx43 has been proposed to be the effector of fast Vj-dependent gating of Cx43 channels. Although it is thought to interact with the pore-affiliated regions to open or close the pore, it is neither a determinant for slow Vj-dependent gating of Cx43 nor a component of the ion-conducting pathway. Unlike Cx43, the voltage-gating properties of Cx45.6 channels do not appear to involve interactions of the carboxyl terminus with the mouth of the pore. Truncation of the carboxyl tail of the human orthologue of Cx45.6, human Cx50, had no effect on its voltage-gating properties or single channel conductance (Xu et al. 2002). In contrast, replacing the N-terminus of Cx45.6 with the N-terminus of Cx43 completely transformed the voltage-gating properties and single channel conductance of Cx45.6 into those of the ‘tailless’ mutant of Cx43. These results suggest that the N-terminus is a major determinant of the unitary conductance and Vj-gating properties of Cx45.6 channels and may be also involved in determining the slow Vj-gating properties of Cx43 channels.
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References |
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D. Shearer, W. Ens, K. Standing, and G. Valdimarsson Posttranslational Modifications in Lens Fiber Connexins Identified by Off-Line-HPLC MALDI-Quadrupole Time-of-Flight Mass Spectrometry Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1553 - 1562. [Abstract] [Full Text] [PDF]
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 N. S. Heyman and J. M. Burt Hindered Diffusion through an Aqueous Pore Describes Invariant Dye Selectivity of Cx43 Junctions Biophys. J., February 1, 2008; 94(3): 840 - 854. [Abstract] [Full Text] [PDF]
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), Cx43 (•) and Cx45.6-Cx43N (
). Points represent the mean ± S.E.M. The continuous lines represent the best fit of the data to a Boltzmann equation.
), Cx43 (
), Alexa 488 (•), Alexa 594 (