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. 2000 Nov;79(5):2403–2415. doi: 10.1016/S0006-3495(00)76485-X

Reversal of the gating polarity of gap junctions by negative charge substitutions in the N-terminus of connexin 32.

P E Purnick 1, S Oh 1, C K Abrams 1, V K Verselis 1, T A Bargiello 1
PMCID: PMC1301127  PMID: 11053119

Abstract

Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage (V(j)), to which they gate by the separate actions of their serially arranged hemichannels (Harris, A. L., D. C. Spray, and M. V. L. Bennett. 1981. J. Gen. Physiol. 77:95-117). Single channel studies of both intercellular and conductive hemichannels have demonstrated the existence of two separate gating mechanisms, termed "V(j)-gating" and "loop gating" (Trexler, E. B., M. V. L. Bennett, T. A. Bargiello, and V. K. Verselis. 1996. Proc. Natl. Acad. Sci. U.S.A. 93:5836-5841). In Cx32 hemichannels, V(j)-gating occurs at negative V(j) (Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello. 1999. J. Gen. Physiol. 114:339-364; Oh, S., C. K. Abrams, V. K. Verselis, and T. A. Bargiello. 2000. J. Gen. Physiol. 116:13-31). A negative charge substitution at the second amino acid position in the N-terminus reverses the polarity of V(j)-gating of Cx32 hemichannels (Verselis, V. K., C. S. Ginter, and T. A. Bargiello. 1994. Nature. 368:348-351;. J. Gen. Physiol. 116:13-31). We report that placement of a negative charge at the 5th, 8th, 9th, or 10th position can reverse the polarity of Cx32 hemichannel V(j)-gating. We conclude that the 1st through 10th amino acid residues lie within the transjunctional electric field and within the channel pore, as in this position they could sense changes in V(j) and be largely insensitive to changes in absolute membrane potential (V(m)). Conductive hemichannels formed by Cx32*Cx43E1 containing a negatively charged residue at either the 8th or 10th position display bi-polar V(j)-gating; that is, the open probability of hemichannels formed by these connexins is reduced at both positive and negative potentials and is maximal at intermediate voltages. In contrast, Cx32*Cx43E1 hemichannels with negative charges at either the 2nd or 5th positions are uni-polar, closing only at positive V(j). The simplest interpretation of these data is that the Cx32 hemichannel can adopt at least two different open conformations. The 1st-5th residues are located within the electric field in all open channel conformations, while the 8th and 10th residues lie within the electric field in one conformation and outside the electric field in the other conformation.

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Selected References

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  1. Barrio L. C., Suchyna T., Bargiello T., Xu L. X., Roginski R. S., Bennett M. V., Nicholson B. J. Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8410–8414. doi: 10.1073/pnas.88.19.8410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bezrukov S. M., Vodyanoy I. Probing alamethicin channels with water-soluble polymers. Effect on conductance of channel states. Biophys J. 1993 Jan;64(1):16–25. doi: 10.1016/S0006-3495(93)81336-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bruzzone R., White T. W., Scherer S. S., Fischbeck K. H., Paul D. L. Null mutations of connexin32 in patients with X-linked Charcot-Marie-Tooth disease. Neuron. 1994 Nov;13(5):1253–1260. doi: 10.1016/0896-6273(94)90063-9. [DOI] [PubMed] [Google Scholar]
  4. Bukauskas F. F., Elfgang C., Willecke K., Weingart R. Heterotypic gap junction channels (connexin26-connexin32) violate the paradigm of unitary conductance. Pflugers Arch. 1995 Apr;429(6):870–872. doi: 10.1007/BF00374812. [DOI] [PubMed] [Google Scholar]
  5. Bukauskas F. F., Peracchia C. Two distinct gating mechanisms in gap junction channels: CO2-sensitive and voltage-sensitive. Biophys J. 1997 May;72(5):2137–2142. doi: 10.1016/S0006-3495(97)78856-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bukauskas F. F., Weingart R. Voltage-dependent gating of single gap junction channels in an insect cell line. Biophys J. 1994 Aug;67(2):613–625. doi: 10.1016/S0006-3495(94)80521-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen D., Eisenberg R. Charges, currents, and potentials in ionic channels of one conformation. Biophys J. 1993 May;64(5):1405–1421. doi: 10.1016/S0006-3495(93)81507-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harris A. L., Spray D. C., Bennett M. V. Kinetic properties of a voltage-dependent junctional conductance. J Gen Physiol. 1981 Jan;77(1):95–117. doi: 10.1085/jgp.77.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hertzberg E. L., Disher R. M., Tiller A. A., Zhou Y., Cook R. G. 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 Dec 15;263(35):19105–19111. [PubMed] [Google Scholar]
  10. Krasilnikov O. V., Yuldasheva L. N., Nogueira R. A., Rodrigues C. G. The diameter of water pores formed by colicin Ia in planar lipid bilayers. Braz J Med Biol Res. 1995 Jun;28(6):693–698. [PubMed] [Google Scholar]
  11. Moreno A. P., Rook M. B., Fishman G. I., Spray D. C. Gap junction channels: distinct voltage-sensitive and -insensitive conductance states. Biophys J. 1994 Jul;67(1):113–119. doi: 10.1016/S0006-3495(94)80460-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Obaid A. L., Socolar S. J., Rose B. Cell-to-cell channels with two independently regulated gates in series: analysis of junctional conductance modulation by membrane potential, calcium, and pH. J Membr Biol. 1983;73(1):69–89. doi: 10.1007/BF01870342. [DOI] [PubMed] [Google Scholar]
  13. Oh S., Abrams C. K., Verselis V. K., Bargiello T. A. Stoichiometry of transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a connexin32 chimera. J Gen Physiol. 2000 Jul 1;116(1):13–31. doi: 10.1085/jgp.116.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Oh S., Ri Y., Bennett M. V., Trexler E. B., Verselis V. K., Bargiello T. A. Changes in permeability caused by connexin 32 mutations underlie X-linked Charcot-Marie-Tooth disease. Neuron. 1997 Oct;19(4):927–938. doi: 10.1016/s0896-6273(00)80973-3. [DOI] [PubMed] [Google Scholar]
  15. Oh S., Rubin J. B., Bennett M. V., Verselis V. K., Bargiello T. A. Molecular determinants of electrical rectification of single channel conductance in gap junctions formed by connexins 26 and 32. J Gen Physiol. 1999 Sep;114(3):339–364. doi: 10.1085/jgp.114.3.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Omori Y., Mesnil M., Yamasaki H. Connexin 32 mutations from X-linked Charcot-Marie-Tooth disease patients: functional defects and dominant negative effects. Mol Biol Cell. 1996 Jun;7(6):907–916. doi: 10.1091/mbc.7.6.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pfahnl A., Zhou X. W., Werner R., Dahl G. A chimeric connexin forming gap junction hemichannels. Pflugers Arch. 1997 Apr;433(6):773–779. doi: 10.1007/s004240050344. [DOI] [PubMed] [Google Scholar]
  18. Rubin J. B., Verselis V. K., Bennett M. V., Bargiello T. A. Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32. Biophys J. 1992 Apr;62(1):183–195. doi: 10.1016/S0006-3495(92)81804-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Spray D. C., Harris A. L., Bennett M. V. Equilibrium properties of a voltage-dependent junctional conductance. J Gen Physiol. 1981 Jan;77(1):77–93. doi: 10.1085/jgp.77.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Suchyna T. M., Nitsche J. M., Chilton M., Harris A. L., Veenstra R. D., Nicholson B. J. Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels. Biophys J. 1999 Dec;77(6):2968–2987. doi: 10.1016/S0006-3495(99)77129-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Swenson K. I., Jordan J. R., Beyer E. C., Paul D. L. Formation of gap junctions by expression of connexins in Xenopus oocyte pairs. Cell. 1989 Apr 7;57(1):145–155. doi: 10.1016/0092-8674(89)90180-3. [DOI] [PubMed] [Google Scholar]
  22. Trexler E. B., Bennett M. V., Bargiello T. A., Verselis V. K. Voltage gating and permeation in a gap junction hemichannel. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5836–5841. doi: 10.1073/pnas.93.12.5836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Veenstra R. D. Size and selectivity of gap junction channels formed from different connexins. J Bioenerg Biomembr. 1996 Aug;28(4):327–337. doi: 10.1007/BF02110109. [DOI] [PubMed] [Google Scholar]
  24. Verselis V. K., Bennett M. V., Bargiello T. A. A voltage-dependent gap junction in Drosophila melanogaster. Biophys J. 1991 Jan;59(1):114–126. doi: 10.1016/S0006-3495(91)82204-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Verselis V. K., Ginter C. S., Bargiello T. A. Opposite voltage gating polarities of two closely related connexins. Nature. 1994 Mar 24;368(6469):348–351. doi: 10.1038/368348a0. [DOI] [PubMed] [Google Scholar]
  26. Vodyanoy I., Bezrukov S. M. Sizing of an ion pore by access resistance measurements. Biophys J. 1992 Apr;62(1):10–11. doi: 10.1016/S0006-3495(92)81762-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

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