Identification of a Pore Lining Segment in Gap Junction Hemichannels

Overview

Journal Biophys J

Publisher Cell Press

Specialty Biophysics

Date 1997 May 1

PMID 9129799

Citations 54

Authors

X W Zhou

A PFAHNL

R Werner

A Hudder

A Llanes

A Luebke

G Dahl

Affiliations

Soon will be listed here.

Abstract

The ability of certain connexins to form open hemichannels has been exploited to study the pore structure of gap junction (hemi)channels. Cysteine scanning mutagenesis was applied to cx46 and to a chimeric connexin, cx32E(1)43, which both form patent hemichannels when expressed in Xenopus oocytes. The thiol reagent maleimido-butyryl-biocytin was used to probe 12 cysteine replacement mutants in the first transmembrane segment and two in the amino-terminal segment. Maleimido-butyryl-biocytin was found to inhibit channel activity with cysteines in two equivalent positions in both connexins: I33C and M34C in cx32E(1)43 and I34C and L35C in cx46. These two positions in the first transmembrane segment are thus accessible from the extracellular space and consequently appear to contribute to the pore lining. The data also suggest that the pore structure is complex and may involve more than one transmembrane segment.

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References

Haefliger J, Bruzzone R, Jenkins N, Gilbert D, Copeland N, Paul D . Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping. J Biol Chem. 1992; 267(3):2057-64. View

Kumar N, Gilula N . Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J Cell Biol. 1986; 103(3):767-76. PMC: 2114303. DOI: 10.1083/jcb.103.3.767. View

Rahman S, Evans W . Topography of connexin32 in rat liver gap junctions. Evidence for an intramolecular disulphide linkage connecting the two extracellular peptide loops. J Cell Sci. 1991; 100 ( Pt 3):567-78. DOI: 10.1242/jcs.100.3.567. View

Dahl G, Werner R, Levine E, Rabadan-Diehl C . Mutational analysis of gap junction formation. Biophys J. 1992; 62(1):172-80; discussion 180-2. PMC: 1260513. DOI: 10.1016/S0006-3495(92)81803-9. View

Durell S, Guy H . Atomic scale structure and functional models of voltage-gated potassium channels. Biophys J. 1992; 62(1):238-47; discussion 247-50. PMC: 1260525. DOI: 10.1016/S0006-3495(92)81809-X. View

Cowan S, Schirmer T, Rummel G, Steiert M, Ghosh R, Pauptit R . Crystal structures explain functional properties of two E. coli porins. Nature. 1992; 358(6389):727-33. DOI: 10.1038/358727a0. View

Akabas M, Stauffer D, Xu M, Karlin A . Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science. 1992; 258(5080):307-10. DOI: 10.1126/science.1384130. View

Bayer E, Safars M, Wilchek M . Selective labeling of sulfhydryls and disulfides on blot transfers using avidin-biotin technology: studies on purified proteins and erythrocyte membranes. Anal Biochem. 1987; 161(2):262-71. DOI: 10.1016/0003-2697(87)90450-7. View

Beyer E, Paul D, Goodenough D . Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol. 1987; 105(6 Pt 1):2621-9. PMC: 2114703. DOI: 10.1083/jcb.105.6.2621. View

10.

Milks L, Kumar N, Houghten R, Unwin N, Gilula N . Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations. EMBO J. 1988; 7(10):2967-75. PMC: 454678. DOI: 10.1002/j.1460-2075.1988.tb03159.x. View

11.

Goodenough D, Paul D, Jesaitis L . Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions. J Cell Biol. 1988; 107(5):1817-24. PMC: 2115343. DOI: 10.1083/jcb.107.5.1817. View

12.

Hertzberg E, Disher R, Tiller A, Zhou Y, Cook R . Topology of the Mr 27,000 liver gap junction protein. Cytoplasmic localization of amino- and carboxyl termini and a hydrophilic domain which is protease-hypersensitive. J Biol Chem. 1988; 263(35):19105-11. View

13.

Yancey S, John S, Lal R, Austin B, REVEL J . The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains. J Cell Biol. 1989; 108(6):2241-54. PMC: 2115622. DOI: 10.1083/jcb.108.6.2241. View

14.

Horton R, Cai Z, Ho S, Pease L . Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques. 1990; 8(5):528-35. View

15.

Unwin N . The structure of ion channels in membranes of excitable cells. Neuron. 1989; 3(6):665-76. DOI: 10.1016/0896-6273(89)90235-3. View

16.

Yellen G, Jurman M, Abramson T, Mackinnon R . Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel. Science. 1991; 251(4996):939-42. DOI: 10.1126/science.2000494. View

17.

Bennett M, Barrio L, Bargiello T, Spray D, Hertzberg E, Saez J . Gap junctions: new tools, new answers, new questions. Neuron. 1991; 6(3):305-20. DOI: 10.1016/0896-6273(91)90241-q. View

18.

Dahl G, Levine E, Rabadan-Diehl C, Werner R . Cell/cell channel formation involves disulfide exchange. Eur J Biochem. 1991; 197(1):141-4. DOI: 10.1111/j.1432-1033.1991.tb15892.x. View

19.

John S, REVEL J . Connexon integrity is maintained by non-covalent bonds: intramolecular disulfide bonds link the extracellular domains in rat connexin-43. Biochem Biophys Res Commun. 1991; 178(3):1312-8. DOI: 10.1016/0006-291x(91)91037-d. View

20.

Paul D, Ebihara L, Takemoto L, Swenson K, Goodenough D . Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell Biol. 1991; 115(4):1077-89. PMC: 2289939. DOI: 10.1083/jcb.115.4.1077. View