Chick Connexin-56, a Novel Lens Gap Junction Protein

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 1, Issue of January 5,5pp: 706-712,1993 ranted m U.S.A.

Chick Connexin-56, a Novel Lens Gap Junction Protein MOLECULAR CLONING AND FUNCTIONAL EXPRESSION*

(Received for publication, August 5, 1992)

Diane M. Rup$, Richard D. Veenstrasll, Hong-Zahn Wangs, Peter R. Brink11 , and Eric C. Beyer$lI**$$§# From the Departments of $Pediatrics, **Medicine, and $$Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, the $Department of Pharmacology, State University of New York Health Science Center, Syracuse, New York 13210, and the IlDepartrnent of Physiology and Biophysics, State University of New York Health Science Center, Stony Brook, New York 1 1 794

We used primers corresponding to the amino-terminal sequence shared by rat connexin-46 and ovine MP70 and a consensus sequence of the second extracellular loop conserved in all connexins to amplify and subsequently clone from chick genomic DNA a new member of the connexin family of gap junction proteins, chick connexin-56. The derived chick connexin- 56 polypeptide contains 510 amino acids with a predicted molecular mass of 55,857 daltons. Although identical in the first 70 amino acids to rat connexin- 46, chick connexin-56 diverges significantly in length and composition in predicted cytoplasmic regions, which have previously been inferred to determine functional and regulatory specificity. We were able to detect hybridization of connexin-56 probes only to RNA derived from lens. Connexin-56 was functionally expressed by the stable transfection of communication- deficient Neuro2A cells. The connexin-56-transfected cells demonstrated intercellular coupling by transfer of microinjected 6-carboxyfluorescein. Double whole- cell patch clamp recordings demonstrated electrical coupling. The induced intercellular conductances were insensitive to uncoupling by heptanol, octanol, or acidification. This behavior of chick connexin-56 may explain previous observations of the unusual physiology of lens fiber gap junctions.

Gap junctions are membrane specializations found between adjacent cells in most tissues, which contain channels provid- ing a low resistance pathway for the intercellular exchange of ions and small molecules. These channels are formed by members of a family of proteins known as connexins, which contain highly conserved extracellular and transmembrane domains, but differing cytoplasmic regions (Beyer et al., 1990).

* These studies were supported by National Institutes of Health Grants HL45466 (to E. C. B. and R. D. V.), EY08368 (to E. C. B.), EY06381 (to D. M. R.), HL 31299 (to P. R. B.) and HL42220 (to R. D. V.) and by grants from the American Heart Association (Grant CSA870405 to E. C. B.) and the McDonnell Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(sl L02838.

1 Established Investigators of the American Heart Association. $§ To whom correspondence should be addressed Div. of Pediatric

Hematology/Oncology, Box 8116, Washington University School of Medicine, 400 S. Kingshighway Blvd., St. Louis, MO 63110. Tel.: 314- 454-2492; Fax: 314-454-2685.

These variable domains, which identify unique members of the connexin family, may confer different physiological properties and thereby contribute to tissue-specific gap junction function. Electrophysiological studies have demonstrated that the gap junctions between cells isolated from different sources differ in a number of properties including unitary conductance and sensitivity to voltage, pH, and various pharmacologic agents (reviewed by Bennett et al., 1991). Functional expression of cloned connexin DNAs in Xenopus oocytes or transfected cells has confirmed that different connexins form channels with different unitary conductances and voltage dependence (Moreno et al., 1991a, 1991b; Veenstra et al., 1993; Hennemann et al., 1992).

The lens is an avascular organ which depends on an exten- sive system of gap junctions for tissue function and homeo- stasis (Goodenough, 1979). This network of gap junctions connects the differentiated lens fiber cells with each other and with the surface epithelium. During differentiation, in- terior fiber cells become metabolically insufficient with the loss of intracellular organelles, mitochondria, and nuclei, and become increasingly distant from the nutrient supply of the aqueous humor. Metabolic coupling provided by gap junctions may compensate for the lack of a blood supply, facilitate strict cellular volume control, and maintain the small intercellular distances which allow for lens transparency (Mathias et al., 1985).

Gap junctions are present at distinct histological locations in the lens tissue, and these different junctions likely correspond to different channel proteins with different properties. They appear morphologically different; freeze fracture studies have demonstrated differences in appearance of fiber-fiber and epithelial-epithelial gap junctions (Miller and Gooden- ough, 1986). They appear physiologically different; in dye transfer experiments, lens fiber junctions appear resistant to uncoupling by acidification, whereas the epithelial-epithelial cell junctions (and most other described gap junctions) demonstrate reversible uncoupling by similar treatment (Miller and Goodenough, 1986; Schuetze and Goodenough, 1982).

We have sought to identify the proteins that form lens gap junctions in order to investigate their functions and regula- tion. The gap junctions between epithelial cells are composed of connexin-43 (Cx43)’ (Musil et al., 1990). Cx43 is also expressed in many other locations, including the heart, and

The abbreviations used are: Cx, connexin; bp, base pairs; kb, kilobase pairs; PCR, polymerase chain reaction; VI, holding potential of cell 1; V,, holding potential of cell 2; V,, transjunctional potential; 11, holding current of cell 1; I,, holding current of the nonpulsed cell (cell 2); I,, junctional current; g,, junctional conductance; 6-CF, 6- carboxy fluorescein; pS, picosiemens; nS, nanosiemens.

706

Chick Connexin-56 707

its biochemical and biophysical properties have been well studied.

In contrast, the gap junctions between fiber cells are less well understood. Connexin proteins have been identified in these cells. Kistler et al. (1985) used a monoclonal antibody to identify a 70-kDa component of sheep lens fiber gap junctions which he called MP70. To date, MP70 has not been cloned; but, the sequence of the 20 amino-terminal amino acids in this protein demonstrate its homology with other connexins. Recently, Paul et al. (1991) cloned a connexin sequence from a rat lens cDNA library called Cx46. While Cx46 shares the same first 20 amino acids with MP70, and anti-Cx46 antisera react with rat lens fiber gap junctions, immunoblotting experiments suggest that Cx46 and MP70 are, in fact, distinct proteins (Paul et al., 1991).

We have sought to further our studies of chicken lens gap junctions by identifying a chick lens fiber connexin. The chicken lens has significant advantages for physiological, developmental, and tissue culture studies (Schuetze and Goodenough, 1982; Menko et al., 1984, 1987). In this paper, we report the molecular cloning and characterization of a new member of the gap junction family of proteins, chick connexin-56 (Cx56). The primary sequence of chick Cx56 demonstrates its close relation to MP70 and rat Cx46. We have only detected Cx56 in lens RNA, not RNA from other sources. We have demonstrated by transfection of a communication- deficient cell line that Cx56 can form intercellular channels; however, Cx56-expressing cell pairs are resistant to some common gap junction uncouplers, suggesting a molecular ex- planation for the atypical physiological behavior of lens fiber gap junctions.

MATERIALS AND METHODS

PCR Amplification of Initial Cx56 Frugment-We used the PCR

sequences shared by Cx46 and MP70 (sense) (AGAATTCGCAG- (Saiki et al., 1988) with primers corresponding to the amino-terminal

GAGCACTCTACAGTCAT) and an antisense primer corresponding to the second extracellular loop conserved in all connexins (AGCAT- GATGATCATGAAGA/GGT/TCNGTGGG) to amplify chicken genomic DNA. Restriction sites (EcoRI and BclI, respectively) were incorporated into the primers to facilitate subcloning into Bluescript plasmids (Stratagene, San Diego, CA). Each PCR cycle consisted of denaturation at 94 "C for 1 min, annealing at 55 "C for 2 min, and extension at 72 "C for 3 min; a total of 30 cycles were performed.

Library Construction, Screening, and Sequencing-A chicken subgenomic library was constructed by size selecting a fraction of -4 kb from an EcoRI digest of chicken genomic DNA by agarose gel electrophoresis, which was cloned into X ZAP (Stratagene, San Diego, CAI. This subgenomic library was screened by hybridization with the DNA fragment of chick Cx56 according to Beyer et al. (1987) to isolate genomic clones containing the entire coding sequence.

DNA sequencing was performed using plasmid templates, Sequen- ase enzyme (U. S. Biochemical Corp.), and oligonucleotide primers as previously described (Beyer, 1990). Both strands of the full length coding sequence of chick Cx56 were fully sequenced. DNA sequence acquisition and analysis and protein sequence alignments and com- parisons were performed using Microgenie (Beckman Instruments, Palo Alto, CA) software running on an IBM-compatible microcom- puter (Queen and Korn, 1984).

DNA and RNA Blots-Chicken genomic DNA was isolated from blood cells (Gustafson et al., 1987) or was obtained commercially (Clontech). The genomic DNA was digested with restriction enzymes, electrophoresed in 1% agarose gels, and transferred to nylon membranes as previously described (Beyer, 1990).

Total cellular RNA was prepared from cells or tissues according to Chomczynski and Sacchi (1987). Most tissues were obtained from normal chick embryos or adult chickens. Heart tissue was also obtained from adult dogs. Uteri were obtained from Ig-day pregnant rats. RNA was separated on formaldehyde/agarose gels and transferred to nylon membranes as previously described (Beyer et al., 1987). Hybridization was performed using specific 32P-labeled DNA probes prepared using random hexanucleotide primers and the Kle-

now fragment of DNA polymerase I (Feinberg and Vogelstein, 1983; Beyer, 1990).

Cell Cultures-Mouse Neuro2A (N2A) neuroblastoma cells were obtained from the American Type Culture Collection. N2A cells were grown in minimal essential medium (GIBCO-Bethesda Research Laboratories (BRL)) supplemented with 10% heat-inactivated (56 "C for 30 min) fetal calf serum (JRH Biosciences, Lenexa, KS), 1 X nonessential amino acids (GIBCO/BRL), 2 mM L-glutamine, and 100 units/ml penicillin and 100 pg/ml streptomycin (GIBCO/BRL). Prep- aration of N2A cells stably transfected with chick Cx43 has been described (Veenstra et al., 1992).

Connexin-56 Transfection-The full-length coding sequence of chick Cx56 was subcloned into the EcoRI site of pSFFV-neo (Fuhl- bridge et al., 1988). N2A cells in 60-mm dishes were transfected with 20 pg of linearized plasmid using the lipofectin reagent (GIBCO- BRL) according to the manufacturer's directions, and stable, neo- mycin-resistant colonies were selected in 0.5 mg/ml G418 (GIBCO- BRL). Connexin expression was verified by Northern blotting of total RNA prepared from selected clones.

Electrophysiological Studies-Cx56-induced coupling in the transfected N2A cells was studied by double whole cell recording proce- dures identical to those described previously (Veenstra, 1990). Patch electrodes had resistances of 2-4 MQ when filled with a solution containing 100 mM potassium glutamate, 15 mM NaCl, 1 mM KHzP04, 4.6 mM MgClZ, 0.68 mM CaClZ, 5 mM EGTA, 3 mM Na2ATP, 3 mM NaZ phosphocreatine, 25 mM Hepes, pH 7.1. The cells were bathed in a solution containing 142 mM NaCl, 1.3 mM KCl, 0.8 mM MgS04, 0.9 mM NaHZPO4, 1.8 mM CaC12, 5.5 mM dextrose, 10 mM Hepes, pH 7.2. All experiments were performed at room temperature (20-22 "C). Transjunctional voltages of 40 or 80 mV were elicited by stepping the holding potential of cell 1 (Vl) from a common value ( Vl = Vz = -40 mV) to -80 or -120 mV. Five-s duration pulses were applied at a rate of four per min, except when channel activity was observed and pulse durations were increased to 10 s or longer. Trans- junctional potential was assumed to be equal to the difference between the two holding potentials (V, = V, - Vl) and junctional conductance was calculated accordingly (gj = Z,/V,). Errors in gj measurements increase proportionally with g, ; they are 4 0 % for g,<5 nS, but can exceed 50% for gj>20 nS, depending on electrode access resistances.

Dye Transfer Studies-6-Carboxyfluorescein (6-CF, M, 376) was purchased from Molecular Probes (Eugene, OR), was dissolved in 120 mM potassium citrate (pH 9.0) to yield a final stock concentration of 20 mM (6-CF), and was titrated to pH 7.0. Stock 6-CF solution was stored in the dark at -20 "C. Microelectrode tips were backfilled with stock dye solution, and transfected N2A cells were impaled. The fluorescent probe filled the cell by simple diffusion. Typical filling times ranged from 30 s to 2 min. Impalements were monitored on the stage of an inverted microscope (Nikon Diaphot) by low light phase contrast and epifluorescent imaging. Epifluorescence illumination was provided by a 100-watt mercury lamp power source and a 490- nm excitation filter. Photomicrographs were recorded on a Nikon 35- mm camera body attached to the microscope's camera port.

RESULTS

Cloning of Chick Connexin-56"All connexins studied to date lack introns in their coding sequence (Miller et al., 1988; Fishman et al., 1990) and contain highly conserved sequences corresponding to four transmembrane and two extracellular domains within the proteins (Beyer et al., 1990). In addition, the two candidate lens fiber junction proteins, MP70 and rat Cx46, share amino-terminal sequence identity. We took ad- vantage of these characteristics and used the PCR to obtain a fragment of a related, but novel connexin sequence from chicken genomic DNA. Using oligonucleotide primers corresponding to a shared sequence (encoding the amino acids ENAQEHSTVI) near the amino terminus of MP70/Cx46 and to the conserved second extracellular loop, a single 654-bp fragment of DNA was amplified, subcloned, and sequenced. (The PCR amplification strategy is illustrated in Fig. 1.)

The PCR-amplified sequence corresponds to nucleotide residues 35-689 in Fig. 2. This sequence contains a single open reading frame which encodes the conserved transmembrane and extracellular sequences characteristic of the connexin family but also contains a cytoplasmic loop which is unique in length and sequence.

708 Chick Connexin-56

FIG. 1. Strategy for PCR amplification of initial fragment of chick Cx56. Model of connexin membrane topology adapted from Beyer et al. (1990) shows conserved regions as umhaded areas and unique cytoplasmic domains as shaded areas A and B. PCR primers 1 and 2 were chosen to correspond to a region near the amino terminus of MP70/Cx46 and to a conserved region in the second extracellular loop.

Southern blots of chicken genomic DNA digested with a panel of restriction enzymes were hybridized with the 654-bp probe. These blots suggest that this probe identifies a unique, single copy gene (Fig. 3). Comparison to similar Southern blots performed using probes for previously identified chick

B E H P

-23 -9.4 -6.6 -4.4

-2.3 -2.0

FIG. 3. Southern blot analysis of chicken genomic DNA. DNA was digested with BamHI ( B ) , EcoRI ( E ) , HindIII ( H ) , or PstI (P) as indicated, separated by agarose electrophoresis, and transferred to nylon membranes. The blot was hybridized with a DNA probe corresponding to the initial 654-bp PCR fragment (residues 35-689 in Fig. 2). Bands detected are consistent with sites predicted from sequence. PstI cuts a t a site in the middle of the probe sequence to give two detectable fragments. X phage DNA digested with HindIII was used as molecular mass standards with size in kilobases indicated to the right of the blot. For all enzymes shown, hybridization produced only one band. The arrow indicates the 4-kb EcoRI fragment which hybridized. This region was used to construct the subgenomic library.

connexins (Beyer, 1990) demonstrates that this is a distinct gene. In order to obtain the full-length coding sequence for this novel connexin, we constructed a subgenomic library using the 4-kb DNA products of an EcoRI digest of chick genomic DNA which contained DNA hybridizing to this probe (arrow in Fig. 3). The subgenomic library was screened by hybridization with the 654-bp probe under high stringency conditions. A single positive clone was isolated and sequenced.

The isolated clone contains a single open reading frame encoding a polypeptide with the conserved regions characteristic of a connexin. The nucleotide sequence of the predicted translated region is shown in Fig. 2. This sequence predicts a protein of 510 amino acids and a molecular weight of 55,857, which we have termed chick connexin-56 (Cx56). Several observations suggest that this represents the true and com- plete coding sequence of Cx56. Regions 5' and 3' to that shown were partially sequenced and contain multiple termi- nation codons in all reading frames. The presumptive ATG translational start codon is contained within a suitable consensus context (Kozak, 1989) for the initiation of translation and encodes a protein whose amino terminus is very similar to other connexins (see Fig. 4). All of the genomic sequence shown is contained within the expressed Cx56 mRNA, since probes generated by PCR amplification of different regions within this coding sequence all hybridize to an identical 8-kb species in blots of lens RNA.

Comparison of Cx56 to the amino acid sequences of rat Cx46, a lens fiber connexin, and chick Cx43, the lens epithelial cell connexin, demonstrates that this protein is a member of the connexin family with the conservation of the four trans-


Chk Cx56 241 ~ E l Y H L G W K K L K O G m T s q y s l ~ m p v t t l t P v n v t G e s K P v s i . n P p a p p v L . v t ~ t A ~ A p v l ? d ~ r ~ V r p : ~ ~ ? ~ . ~ t ~ a ? ? ? Rat Cx46 225 N!.~IYi;i.G!~KKl,KU(;VT---nh:n------?dasccrhK?--L~?is~-------l:-AnS~l~~s"V.Sia:.-?----?v~ Chk Cx43 225 ~i:lfyvf:KavK::rVk---qitd------i'ys~sG'.msI'--s~dcas-------?-kya~y~~--c~s?~;,-----~~~s

Chk Cx56 371 a a a a P r t r P ~ S n t a S m a S y P v A P ~ v P e n r h r A v t ~ T ~ V s ~ ~ V T i q r ~ y ~ ~ ~ t P a ~ i n y ~ n s ~ r ~ ~ A a r Q ~ ~ v N m a ~ E Q Q g R a t Cx46 279 th--Pac-Pt-vqgkatqfPqA?ll?-----Ad-~T-V---V~~ndaqqrq~Pv-kh-cnqhh-~:~cON~~~AsigA~~C: Chk Cx43 279 p m - s P - - - P g y k l y t q d r n n s s c r n y - - - - - - n k - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ~ A s E ~ ~ ' , ~ A ~ y s ~ ~ ~ z r

Chk Cx56 401 k a P s S S a q S S t ~ S S v r h ~ l p e q e F . P l e ~ l l ~ l ~ a G ~ ~ l t T t n S G S S t s ~ S C ~ ~ s q s k ~ d v f ~ T ; e e L ~ . r ~ ~ ~ ~ s a t c T ~ V ~ W Rat Cx46 342 - - P A S k - p S S a a S S - - - - - - - - - - P " h - - h - - - g ~ k G - - l - T d s S G S S l e c S - ~ ~ v v - - t ~ ~ ~ ~ a a L A - - - - - - - - - ~ T V ~ W C!tk Cx43 336 m ~ q A q S t l S m - s h a - a p f d f a - ~ E h - - Q n t k ~ ~ - - - - - - - - - - - - - - - ~ ~ r ~ l c l a - - D l - - - ~ ~ v ~ - - - - - - - - - - a r - - 6. _j. . ,

Chk CxS6 181 Hr?PPI,lvdtrrlSRASKSSSS~~sDDLAv Rat Cx46 389 t !SPPI .v l ld-Pr -RsSKSSSq~R?qDLAi Chk Cx43 362 ----------PpS~,SsraSSR?R?I)~t.e?

FIG. 4. Comparison of chick Cx56 to several closely related connexins. The derived amino acid sequences of chick Cx56 and those of rat Cx46 (Paul et al., 1991) and chick Cx43 (Musil et al., 1990) are shown as optimally aligned. Residues that are identical to their counterparts in chick Cx56 are represented in uppercase letters; nonidentical residues are listed in lowercase letters. Dashes represent spaces added to optimize alignment. The four putative transmembrane domains in the connexins are marked with long lines above and below. The 3 cysteines uniformly conserved in the two connexin extracellular loops are indicated with the short double lines above and below.

a b c d e f g h i

7.4- 5.3-

0

2.8-

1.9- 1.6-

FIG. 5. RNA blots demonstrating tissue distribution of expression of Cx56 mRNA. Total cellular RNA was prepared from cells or tissues, separated on formaldehyde/agarose gels (10 pg/lane), and prepared for Northern blotting as described under "Materials and Methods." Blots were then incubated with specific 32P-labeled DNA probes for Cx56. Migration of RNA standards (in kilobases) is indicated to the left of the figure.

membrane domains and two extracellular domains (Fig. 4). Chick Cx56 is more similar to rat Cx46 than any other known connexin; their first 70 amino acids are identical, and 62% of amino acids are identical in these two proteins. In contrast, chick Cx56 exhibits only 49% identity to chick Cx43 and 46% identity to mouse Cx50 (White et al., 1992). However, chick Cx56 diverges almost totally from rat Cx46 in predicted cytoplasmic domains. The amino-terminal 20 amino acids of ovine MP70 are also identical to these positions in chick Cx56 (Kistler et al., 1988). Chick Cx56 is the largest connexin polypeptide yet identified, with a cytoplasmic loop 16 amino acids longer than Cx43 or Cx46, but 8 amino acids shorter than Cx45. The carboxyl-terminal tail is also quite long. The carboxyl-terminal region of the protein contains serine-rich regions which, as in other connexins, could serve as substrates for protein phosphorylation. This region and the sequence ARQNWVNMAEQQ (residues 387-399), which has no known function, are relatively conserved in the other proteins

12 16 19 Ad 12 16 19 Ad

Cx56 Cx43 FIG. 6. Northern blots comparing expression of Cx56 and

Cx43 in RNA derived from lenses of chick embryos of different ages and adult chickens. 10 pg of total RNA prepared from lenses from 12-, 16-, or 19-day chick embryos or from adult chickens was applied in each lane and hybridized with probes for Cx56 or Cx43. Arrowheads indicate the migration of 18 S and 27 S rRNAs.

which Bennett et al. (1991) have termed type I1 connexins. RNA blots probed a t high stringency with a Cx56 probe

identified an mRNA of approximately 8-kb which was expressed only in chick lens and not in any other tissue tested (Fig. 5). In addition, expression of the Cx56 mRNA was found to change with development, with an increase in RNA levels between 12- and 19-day-old embryos (Fig. 6). Levels of chick Cx43, the lens epithelial/epithelial gap junction protein, re- main constant over this time period (Fig. 6).

Functional Expression of Cx56"To verify that Cx56 was capable of forming functional cell-to-cell channels and to study the properties of these channels, we stably transfected the communication-deficient cell line N2A with the full- length coding sequence of Cx56. We have recently used this system to examine the gap junctional channels formed by three other chick connexins (Cx42, Cx43, and Cx45) (Veenstra et al., 1993). The parent N2A cell line shows no detectable gap junctional channels when screened by double whole cell

710 Chick Connexin-56

FIG. 7. Dye transfer in Cx56 transfected cells. A, phase contrast image of a field of Cx56-transfected N2A cells. The cell in the center of the field (arrow), in direct apposition to two other cells was injected with 6-carboxyfluorescein. Scale bar, 90 pm. B, epifluorescence micrograph of the same cell field showing the diffusional gradient of dye transfer from the primary injected cell (arrow) to one of two adjacent cells. The photographic image was taken 1 min after removal of the dye electrode, injection time was 30 s. Similar dye coupling was observed in 13 of 51 injected cells.

i

0 <I 1 - 1 0 10-70 >?I)

,Junctional Conductance (nS)

FIG. 8. Junctional conductances of Cx56-transfected cell pairs. The frequency histogram displays junctional conductances for all 66 Cx56 N2A cell pairs examined using conventional double whole cell recording techniques. Transjunctional potentials were 40 or 80 mV. Bin widths of 10 nS were arbitrarily assigned with the exception of gj < 1 nS to illustrate the occurrence of one low conductance cell pair where channel activity was observed. Above 20 nS, gj measurements are severely limited by recording electrode resistances, so no attempt was made to distinguish between gj > 20 nS.

patch clamp recordings and shows no detectable expression of any known connexin (Veenstra et al., 1993). Connexin- transfected N2A clones were selected in G418. RNA blot analysis of total RNA prepared from the transfected cells demonstrated that they expressed Cx56 mRNA, while no hybridization of a Cx56 probe was detected in cells transfected with vector alone (data not shown).

Clones testing positive for connexin expression were ex-

amined for functional coupling by the microinjection of the junction-permanent dye 6-carboxyfluorescein. We observed intercellular passage of the dye in 25% of the transfected cell injections (13 of 51 trials, Fig. 7). The intercellular transfer appeared consistent with the slow diffusion of dye through gap junctions as evidenced by the gradient of intensity between the injected cell and its coupled neighbors and a gradual increase in intensity in the coupled cell demonstrated by serial observations.

The Cx56-transfected cells were also examined by using the double whole cell patch clamp recording technique. Electrical communication was evident in 40% of pairs examined (see Fig. 8), whereas untransfected cells showed no coupling. The distribution of junctional conductances ( gj) from all 66 pairs examined (Fig. 8) revealed a high incidence of well coupled (25%, gj > 20 nS) and uncoupled (59%, gj = 0 nS) cell pairs. This distribution is quite different from what we have observed for other Cx-transfected N2A cell pairs, where gjs of 3-6 nS were most frequently observed (Veenstra et al., 1993). In one poorly coupled cell pair ( gj < 1 nS), quantal changes in junctional current ( Z j ) , indicative of channel activity, were apparent (Fig. 9). Channel currents were obtained a t four different transjunctional voltages, and the mean channel conductance ranged from 96 to 100 pS. Linear regression analysis of the mean single channel currents from +40 to +80 mV produced a slope of 96.5 pS.

In those pairs where gj was less than 20 nS and our methods are more accurate for determining gj, we attempted to inhibit gj pharmacologically by superfusion with 2 mM 1-heptanol or 1-octanol and 50 mM sodium acetate (sodium acetate) saline buffered to pH 5.0-6.0. In chick Cx43-transfected N2A cell pairs, treatment with 2 mM heptanol or pH 6.0 sodium acetate saline completely inhibited gi. Similar treatments applied to Cx56-transfected N2A cell pairs failed to produce any inhibition of gj in five attempts. Stronger uncoupling treatments with 2 mM octanol or pH 5.0 sodium acetate saline also failed to produce any inhibition of gi in Cx56-transfected cells (Fig. 10). Also in contrast to our findings with four other connexins (Veenstra et al., 1992),* no voltage dependence of gj was observed with transjunctional voltages up to k 100 mV in Cx- 56-transfected cell pairs. These findings suggest that the physiological modulation of Cx56 is quite different from those connexins expressed in other tissues.

DISCUSSION

We have reported the molecular cloning and functional characterization of a novel gap junction protein, chick Cx56. The primary sequence of Cx56 establishes that this protein is a member of the connexin family of gap junction proteins with conserved regions corresponding to the four transmembrane and two extracellular domains (Beyer et al., 1990). The sequence corresponding to the predicted cytoplasmic domains is longer and distinct from any previously studied connexin, thus identifying Cx56 as a new and unique member of this family.

We have shown in RNA blots that Cx56 mRNA is expressed exclusively in the lens. Cx43 was previously identified as a component of lens epithelial gap junctions (Musil et al., 1990). I t is possible that Cx56 is the chick lens fiber connexin, as suggested by its abundance in the lens and its striking homology to the amino-terminal regions of ovine MP70 and rat Cx46, two known components of the lens fiber junction (Paul et al., 1991). Indeed, it is possible that Cx56 is the chicken homolog of MP70; but, we have been unable to test this possibility because the monoclonal antibodies of Kistler et al.

* K. E. Reed, E. M. Westphale, D. M. Larson, H.-Z. Wang, R. D. Veenstra, and E. C. Beyer, manuscript submitted.


A m

Cx56

......

..... .

0 1 2 3 4 5 6 7

Time (Sec)

C Cx56

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B Cx56

x n I i "- 3001 Ill. I

250

2 0 0 ~ -

150"

1 0 0 ~ ~

50.-

n- I -5 0 5 10 15 20

Junctional Current (PA)

Cx56

Channel Conductance (pS) 30 40 50 60 70 80 Transjunctional Voltage (mV)

FIG. 9. Junctional channel currents and conductance of one Cx66-transfected N2A cell pair. A , paired whole cell currents from cell 1 and 2 (Zl and Z2) of a Cx56-transfected cell pair. A -50-mV voltage step was applied to cell 1 at time zero and junctional currents appear as quantal fluctuations of equal amplitude and opposite polarity. The arrows indicate the deletion of 170 pA of nonjunctional current ( A ) associated with the VI voltage step and a 4.2-5 interval of the continuous current records ( B ) for display purposes only. Both currents were low pass filtered at 125 Hz and digitally sampled at 1 kHz. B, frequency histogram of all 25,928 digitized points representing the entire Z2 ( I j ) current trace from A. The area under each gaussian peak is proportional to the time spent in each state, and the mean (solid l i n e ) of each peak indicates the current amplitudes for the observed channel activity. Channel conductances were calculated by dividing the mean current amplitude by the transjunctional potential of 50 mV. Transitions between adjacent states (channel events) were counted by setting threshold discriminators to the minimum between adjacent peaks and scanning the Z2 trace for event crossings with a minimum duration of 1 ms. C, event amplitude histogram for all 752 channel events. Event conductances are normally distributed around a mean f S.D. of 96 f 6 pS (for this example at 50 mV). D, mean f S.D. channel current for all events obtained from four different transjunctional potentials. Event counts were 951, 752, 524, and 262 for 40, 50, 60, and 70 mV, respectively. The solid l ine represents the result of linear regression analysis of the channel current-voltage plot.

(1985) show no reactivity with chicken l e n ~ e s . ~ I t is also possible that Cx56 is expressed in lens epithelial cells or, indeed, in both lens cell types. Definitive localization will require immunocytochemistry using an anti-Cx56 antibody. White et al. (1992) have recently cloned another connexin (mouse Cx50) which is immunologically related to MP70. This sequence is quite different from chick Cx56 suggesting an increased complexity or variation among lens connexins.

The 8-kb mRNA encoded by Cx56 is much longer than is required for encoding a protein of 55,857 daltons, and suggests the presence of unusually long 5'- and 3"untranslated regions. Previously examined connexin cDNAs contain no more than -2 kb of untranslated sequence. However, small coding regions surrounded by large noncoding regions have been observed in a small subset of other genes, including bcl-2 and other cellular oncogenes (Negrini et al., 1987) whose expression is tightly regulated. These untranslated regions have been implicated in the translational control of expression (Kozak, 1986). Such controls may be of unique importance in the lens fiber cell which becomes progressively restricted in its biosynthetic capacity. The biologic significance of these regions in chick Cx56 could be addressed by further molecular characterization of Cx56 cDNAs and in vitro transcription and translation of constructs containing translated and un-

D. M. Rup and E. C. Beyer, unpublished observations.

translated Cx56 regions in the context of a reporter gene. Our functional expression of chick Cx56 by the stable

transfection of N2A cells demonstrates thet this sequence is indeed capable of facilitating intercellular dye passage and electrical coupling as would be expected for a gap junction protein. We have also rarely observed single channel events characteristic of gap junctional channels. Indeed, Cx56 channels differ in unitary conductance from any previously char- acterized cloned connexins (Fishman et al., 1990; Moreno et al., 1991b; Veenstra et al., 1992).

However, the electrophysiologic data suggest that the intercellular communication induced by Cx56 differs from that observed with other previously expressed connexins. Cx56- induced coupling appears resistant to uncoupling with lipo- philic agents such as heptanol or by cytoplasmic acidification. However, there are some precedents for similar gap junction behavior. Bukauskas et al. (1992) have studied an arthropod gap junction that appears insensitive to lipophiles. Previous dye transfer studies in whole chick lens have documented the presence of two physiologically distinct gap junctions (Miller and Goodenough, 1986); acidification by treatment with a 90% Cop-equilibrated medium blocked intraepithelial dye transfer but did not affect fiber-to-fiber cell transfer. The dye transfer between fiber cells of the embryonic chick lens was unaffected by COz treatment or acetate treatment beyond

712 Chick Connexin-56 Cx56

600 100 B c 400

U r 200

c 60 U

r I. 20 e 0 n e t -200

" -20

t

-400

-600

-60

(PA) (PA) 0 1 2 3 4 5 6 7 8

-100 0 1 2 3 4 5 6 7 8

Elapsed Time (Min) Elapsed Time (Min)

Elapsed Time (Min) Elapsed Time(Min)

FIG. 10. Effects on CxS6 and Cx43 junctional conductances of 1-alkanols or low pH. Steady state junctional conductance (g,) was determined from the end of 5-5 pulses to transjunctional potentials (Vi) of +40 mV applied at the rate of four per min. A, paired whole cell currents recorded from one Cx56-transfected N2A cell pair during superfusion with 2 mM octanol. The top truce corresponds to the junctional current (4) measured in the nonpulsed cell, while the currents from the pulsed cell are shown below ( I , - Zj). 9 s of each recovery interval ( v i = 0 mV) between successive pulses were deleted from the current teraces for clarity of presentation. B, paired whole cell currents recorded from one Cx43-transfected N2A cell pair during superfusion with 2 mM heptanol using the identical protocol to that described above. Note the decrease in junctional current during exposure to 2 mM heptanol which was gradually reversible upon washout of the uncoupling agent with normal saline (2 ml/min, 2-ml bath volume). C, effect of 1-alkanol application on gj in Cx43 and Cx56 cell pairs. The time course of the change in gj during exposure to 2 mM octanol or heptanol is shown for Cx56 or Cx43 cell pairs illustrated in A and E . Exposure of five Cx56 cell pairs to 2 mM heptanol (or 2 mM octanol) had no effect on gj, while the same treatment applied to three Cx43 cell pairs reversibly inhibited gj by >95%. D, effect of low pH on gj in Cx43 and Cx56 cell pairs. The time course of the change in gj during exposure to 50 mM sodium acetate/saline (buffered to the indicated pH) is illustrated for Cx43 and Cx56 cell pairs. Exposure of a Cx43 cell pair to pH 6.0 saline reversibly inhibited gj following a somewhat slower time course than inhibition by 1-alkanol treatment. The same treatment applied to five Cx56 cell pairs had no effect on gi. Even further lowering pH to 5.0 (in the Cx56 cell pair shown) failed to significantly alter gj.

developmental stage 14 (Schuetze and Goodenough, 1982). Thus, the pH-sensitive behavior of gap junctions between epithelial cells correlates with the properties of Cx43 which is expressed at that location (Swenson et al., 1989; Werner et al., 1989; Musil et al., 1990), while the acid insensitivity of fiber-fiber cell junctions correlates with the properties of Cx56. Goodenough (1979) speculated that lens fiber cells have adapted their communication system to be insensitive to conditions which normally trigger a low-to-high resistance switch; Cx56 channels may explain this unique physiology.

Acknowledgments-We are grateful to Dr. H. Lee Kanter and Eileen Westphale for helpful discussions and preparation of many of the RNA samples. Mark Chilton provided valuable technical assist- ance with tissue culture of the transfected N2A cells. Sridhar Goli assisted significantly with channel data analysis.

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Chick Connexin-56, a Novel Lens Gap Junction Protein