chick connexin-56, a novel lens gap junction protein
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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-termi- nal sequence shared by rat connexin-46 and ovine MP70 and a consensus sequence of the second extracel- lular loop conserved in all connexins to amplify and subsequently clone from chick genomic DNA a new member of the connexin family of gap junction pro- teins, chick connexin-56. The derived chick connexin- 56 polypeptide contains 510 amino acids with a pre- dicted 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 acid- ification. This behavior of chick connexin-56 may ex- plain 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 prop- erties 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 expres- sion of cloned connexin DNAs in Xenopus oocytes or trans- fected cells has confirmed that different connexins form chan- nels with different unitary conductances and voltage depend- ence (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 corre- spond 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) dem- onstrate 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.
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 junc- tions 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 con- nexin-56 (Cx56). The primary sequence of chick Cx56 dem- onstrates 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 ge- nomic 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