Abstract
1. Hemichannels formed by connexin26 (Cx26) on the horizontal cell dendrites that invaginate cone terminals in the vertebrate retina have been implicated in the feedback mechanism by which horizontal cells regulate transmitter release from cone photoreceptors. However, their membrane properties had not been studied previously, and it was unclear whether they could subserve their purported function at the membrane potentials over which horizontal cells operate.
2. We used the two-electrode voltage clamp technique to record the membrane currents and pharmacological properties of Cx26 hemichannels formed in the Xenopus oocyte expression system.
3. Oocytes expressing Cx26 exhibited large membrane conductances over a broad range of hyperpolarizing and depolarizing membrane potentials, and displayed little evidence of voltage-dependent gating, indicating that the hemichannels are constitutively open. The Cx26-mediated nonjunctional currents were relatively insensitive to quinine, a cinchona alkaloid that opens hemichannels formed by several other connexins. However, the hemichannel currents were blocked by carbenoxolone, a rise in extracellular calcium, or lowering intracellular pH. The currents could also be suppressed by reducing extracellular pH, and by the chloride channel blocker NPPB through its direct interaction with Cx26 hemichannels.
4. These findings provide a basis with which to evaluate the in situ pharmacological studies that attempt to assess the putative role of Cx26 hemichannels in the feedback pathway in the distal retina.
Keywords: connexin26, hemichannels, connexons, membrane currents, gap junctions, retinal horizontal cells, carbenoxolone, NPPB, pH, calcium, quinine
REFERENCES
- Ahmad, S., and Evans, W. H. (2002). Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: Implications for the assembly of gap junctions. Biochem. J.365:693–699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Al-Ubaidi, M. R., White, T.W., Ripps, H., Poras, I., Avner, P., Gomés, D., and Bruzzone, R. (2000). Functional properties, developmental regulation and chromosomal localization of murine connexin36, a gap junctional protein expressed preferentially in retina and brain. J. Neurosci. Res.59:813–826. [DOI] [PubMed] [Google Scholar]
- Arnos, K. S., Israel, J., Wilson, M. P., and Devlin, L. (1992). Genetics of hearing disorders. Clin. Commun. Disord.2:20–34. [PubMed] [Google Scholar]
- Barnes, S., Merchant, V., and Mahmud, F. (1993). Modulation of transmission gain by protons at the photoreceptor output synapse. Proc. Natl. Acad. Sci. U.S.A.90:10081–10085. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baud, C., Kado,R. T., and Marcher,K. (1982). Sodium channels induced by depolarization of theXenopus laevisoocyte. Proc. Natl. Acad. Sci. U.S.A.79:188–192. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beahm, D. L., and Hall, J. E. (2002). Hemichannel and junctional properties of connexin 50. Biophys. J.82:2016–2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cohen-Salmon, M., Ott, T., Michel, V., Hardelin, J.-P., Perfettini, I., Eybalin, M., Wu, T., Marcus, D. C. Wangemann, P., Willecke, K., and Petit, C. (2002). Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death. Curr. Biol.12:1106–1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dascal, N. (1987). The use of Xenopusoocytes for the study of ion channels. CRC Crit. Rev. Biochem.22:317–387. [DOI] [PubMed] [Google Scholar]
- Davidson, J. S., Baumgarten, I. M., and Harley, E. H. (1986). Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid. Biochem. Biophys. Res. Commun.134:29–36. [DOI] [PubMed] [Google Scholar]
- Delmar,M., Morley,G. E., and Taffet, S. M. (1997). Molecular analysis of the pH regulation of the cardiac gap junction protein connexin 43. In Spooner, P.M., Joyner, R.W., and Jalife, J. (eds.), Discontinuous Conduction in the Heart,Futura, Armonk, NY, pp. 203–221. [Google Scholar]
- DeVries, S. H. (2001). Exocytosed protons feedback to suppress the Ca2+ current in mammalian cone photoreceptors. Neuron32:1107–1117. [DOI] [PubMed] [Google Scholar]
- DeVries, S. H., and Schwartz, E. A. (1992). Hemi-gap junction channels in solitary horizontal cells of the catfish retina. J. Physiol.445:201–230. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dixon, D. B., Takahashi, K.-I., Bieda, M., and Copenhagen, D. R. (1996). Quinine, intracellular pH and modulation of hemi-gap junctions in catfish horizontal cells. Vis. Res.36:3925–3931. [DOI] [PubMed] [Google Scholar]
- Dmitriev,A.V., and Mangel, S.C. (2001). Circadian clock regulation of pH in the rabbit retina. J. Neurosci.21:2897–2902. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ebihara, L., Beyer, E. C., Swenson, K. I., Paul, D. L., and Goodenough, D. A. (1989). Cloning and expression of a Xenopusembryonic gap junction protein. Science243:1194–1195. [DOI] [PubMed] [Google Scholar]
- Eckert, R., Donaldson, K., Goldie, K., and Kistler, J. (1998). A distinct membrane current in rat lens fiber cells isolated under calcium-free conditions. Invest. Ophthalmol. Vis. Sci.39:1280–1285. [PubMed] [Google Scholar]
- Eskandari, S., Zampighi, G. A., Leung, D. W., Wright, E. M., and Loo, D. D. (2002). Inhibition of gap junction hemichannels by chloride channel blockers. J. Membr. Biol.185:93–102. [DOI] [PubMed] [Google Scholar]
- Falk, M. M. (2000). Biosynthesis and structural composition of gap junction intercellular membrane channels. Eur. J. Cell Biol.79:564–574. [DOI] [PubMed] [Google Scholar]
- Frenz, C. M., and van deWater, T. R. (2000). Immunolocalization of connexin 26 in the developing mouse cochlea. Brain Res. Rev.32:172–180. [DOI] [PubMed] [Google Scholar]
- George, C. H., Kendall, J. M., and Evans, W. H. (1999). Intracellular trafficking pathways in the assembly of connexins into gap junctions. J. Biol. Chem.274:8678–8685. [DOI] [PubMed] [Google Scholar]
- Hirasawa, H., and Kaneko, A. (2003). pH changes in the invaginating synaptic cleft mediate feedback from horizontal cells to cone photoreceptors by modulating Ca2+ channels. J. Gen. Physiol.122:657–671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Janssen-Bienhold,U., Schultz, K., Gellhaus, A., Schmidt, P., Ammermuller, J., and Weiler,R. (2001). Identification and localization of connexin26 within the photoreceptor-horizontal cell synaptic complex. Vis. Neurosci.18:169–178. [DOI] [PubMed] [Google Scholar]
- John, S. A., Kondo, R., Wang, S.-Y., Goldhaber, J. I., and Weiss, J. N. (1999). Connexin-43 hemichannels opened by metabolic inhibition. J. Biol. Chem.274:236–240. [DOI] [PubMed] [Google Scholar]
- Kamermans, M., Fahrenfort, I., Schultz, K., Janssen-Bienhold, U., Sjoerdsma, T., and Weiler, R. (2001). Hemichannel-mediated inhibition in the outer retina. Science292:1178–1180. [DOI] [PubMed] [Google Scholar]
- Kamermans, M., and Spekreijse, H. (1999). The feedback pathway from horizontal cells to cones. A mini review with a look ahead. Vis. Res.39:2449–2468. [DOI] [PubMed] [Google Scholar]
- Kelley, P. M., Harris, D. J., Comer, B. C., Askew, J. W., Fowler, T., Smith, S. D., and Kimberling, W. J. (1998). Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss. Am. J. Hum. Genet.62:792–799. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kelsell, D. P., Dunlop, J., Stevens, H. P., Lench, N. J., Liang, J. N., Parry, G., Mueller, R. F., and Leigh, I. M. (1997). Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature87:80–83. [DOI] [PubMed] [Google Scholar]
- Kikuchi, T., Kimura, R. S., Paul, D. L., and Adama, J. C. (1995). Gap junctions in the rat cochlea: Immunohistochemical and ultrastructural analysis. Anat. Embryol.191:101–118. [DOI] [PubMed] [Google Scholar]
- Kondo, R. P., Wang, S.-Y., John, S. A., Weiss, J. N., and Goldhaber, J. (2000). Metabolic inhibition activates a non-selective current through connexin hemichannels in isolated ventricular myocytes. J. Mol. Cell Cardiol.32:1859–1872. [DOI] [PubMed] [Google Scholar]
- Krafte,D. S., and Kass,R. S. (1988). Hydrogen ion modulation of Ca channel current in cardiac ventricular cells. Evidence for multiple mechanisms. J. Gen. Physiol.91:641–657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lautermann, J., Frank, H.G., Jahnke, K., Traub,O., and Winterhager,E. (1999). Developmental expression patterns of connexin26 and-30 in the rat cochlea. Dev. Genet.25:306–311. [DOI] [PubMed] [Google Scholar]
- Li, H., Liu, T.-F., Lazrak, A., Peracchia, C., Goldberg, G. S., Lampe, P. D., and Johnson, R. G. (1996). Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells. J. Cell Biol.134:1019–1030. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Locke, D., Perusinghe, N., Newman, T., Jayatilake, H., Evans, W. H., and Monaghan, P. (2000). Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland. J. Cell Physiol.183:228–237. [DOI] [PubMed] [Google Scholar]
- Malchow, R. P., Qian, H., and Ripps, H. (1993). Evidence for hemi-gap junctional channels in isolated horizontal cells of the skate retina. J. Neurosci. Res.35:237–245. [DOI] [PubMed] [Google Scholar]
- Malchow, R. P., Qian, H., and Ripps,H. (1994).Anovel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina. J. Gen. Physiol.104:1039–1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Martin, P. E., Blundell, G., Ahmad, G., Errington, R., and Evans, W. H. (2001). Multiple pathways in the trafficking and assembly pf connexin 26, 32 and 43 into intercellular communication channels. J. Cell Sci.114:3845–3855. [DOI] [PubMed] [Google Scholar]
- Miledi, R., Parker, I., and Woodward, R. M. (1989). Membrane currents elicited by divalent cations in Xenopusoocytes. J. Physiol.417:173–195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Musil, L. S., and Goodenough, D. A. (1991). Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junction plaques.J. Cell Biol.115:1357–1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Musil, L. S., and Goodenough, D. A. (1993). Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell74:1065–1077. [DOI] [PubMed] [Google Scholar]
- Oh, S., Abrams, C. K., Verselis, V. K., and Bargiello, T.A. (2000). Stoichiometry of transjunctional voltagegating polarity reversal by a negative charge substitution in the amino terminus of a connexin32 chimera. J. Gen. Physiol.116:13–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parkerson, K. A., and Sontheimer, H. (2003). Contribution of chloride channels to volume regulation of cortical astrocytes. Am. J. Physiol. Cell Physiol.284:C1460–C1467. [DOI] [PubMed] [Google Scholar]
- Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I., and Goodenough, D. A. (1991). Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopusoocytes. J. Cell Biol.115:1077–1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pfahnl, A., and Dahl,G. (1999). Gating of Cx46 gap junction hemichannels by calcium and voltage. Pflugers Arch. Eur. J. Physiol.437:345–353. [DOI] [PubMed] [Google Scholar]
- Pottek, M., Hoppenstedt,W., Janssen-Bienhold,U. Schultz, K., Perlman, I., and Weiler,R. (2003). Contribution of connexin26 to electrical feedback inhibition in the turtle retina. J. Comp. Neurol.466:468–477. [DOI] [PubMed] [Google Scholar]
- Qian, H., Malchow, R. P., and Ripps, H. (1989). A novel voltage-sensitive conductance in skate horizontal cells. Invest. Ophthalmol. Vis. Sci. 30(Suppl.):67. [Google Scholar]
- Qian, H., Malchow, R. P., and Ripps, H. (1993). Gap junctional properties of electrically coupled skate horizontal cells in culture. Vis. Neurosci.10:287–295. [DOI] [PubMed] [Google Scholar]
- Quist, A. P., Rhee, S. K., Lin, H., and Lal, R. (2000). Physiological role of gap-junctional hemichannels: Extracellular calcium-dependent isosmotic volume regulation. J. Cell Biol.148:1063–1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rae, J. I., Dewey, J., and Rae, J. S. (1992). The large-conductance potassium ion channel of rabbit corneal epithelium is blocked by quinidine. Invest. Ophthalmol. Vis. Sci.33:286–290. [PubMed] [Google Scholar]
- Ripps, H., Qian, H., and Zakevicius, J. (2002a). Blockade of an inward sodium current facilitates pharmacological study of hemi-gap-junctional currents Xenopusoocytes. Biol. Bull.203:192–194. [DOI] [PubMed] [Google Scholar]
- Ripps, H., Qian, H., and Zakevicius, J. (2002b). Pharmacological enhancement of hemi-gap-junctional currents in Xenopusoocytes. J. Neurosci. Meth.121:81–92. [DOI] [PubMed] [Google Scholar]
- Spicer, S. S., and Schulte, B. A. (1998). Evidence for a medial K+ recycling pathway from inner hair cells. Hear. Res.118:1–12. [DOI] [PubMed] [Google Scholar]
- Srinivas, M., Hopperstad, M. G., and Spray, D. C. (2001). Quinine blocks specific gap junction channel subtypes. Proc. Natl. Acad. Sci. U.S.A.98:10942–10947. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Srinivas, M., and Spray, D. C. (2003). Closure of gap junction channels by arylaminobenzoates. Mol. Pharmacol.63:1389–1397. [DOI] [PubMed] [Google Scholar]
- Tekin, M., Arnos, K. S., Xia, X. J., Oelrich, M. K., Liu, X. Z., Nance, W. E., and Pandya, A. (2001). W44C mutation in the connexin 26 gene associated with dominant non-syndromic deafness. Clin. Genet.59:269–273. [DOI] [PubMed] [Google Scholar]
- Thoreson,W. B., Bryson, E. J., and Rabl, K. (2003). Reciprocal interactions between calcium and chloride in rod photoreceptors. J. Neurophysiol.90:1747–1753. [DOI] [PubMed] [Google Scholar]
- Tokimasa, T., and North, R. A. (1996). Effects of barium, lanthanum and gadolinium on endogenous chloride and potassium currents in Xenopusoocytes. J. Physiol.496:677–686. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Trexler, E. B., Bennett, M.V. L., Bargiello, T. A., and Verselis,V.K. (1996).Voltage gating and permeation in a gap junction hemichannel. Proc. Natl. Acad. Sci. U.S.A.93:5836–5841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Trexler, E. B., Bukauskas, F. F., Bennett, M. V. L., Bargiello, T. A., and Verselis, V. K. (1999). Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation. J. Gen.Physiol.113:721–742. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Verselis, V. K., Trexler, E. B., and Bukauskas, F. F. (2000). Connexin hemichannels and cell-cell channels: Comparison of properties. Braz. J. Med. Biol. Res.33:379–389. [DOI] [PubMed] [Google Scholar]
- Weber, W.-M. (1999). Endogenous ion channels in oocytes of Xenopus laevis: Recent developments. J. Membr. Biol.170:1–12. [DOI] [PubMed] [Google Scholar]
- White, T. W. (2000). Functional analysis of human Cx26 mutations associated with deafness. Brain Res. Rev.32:181–183. [DOI] [PubMed] [Google Scholar]
- White, T. W., Deans, M. R., O'Brien, J., Al-Ubaidi, M. R., Goodenough, D. A., Ripps, H., and Bruzzone, R. (1999). Functional characteristics of skate connexin35, a member of the ?subfamily of connexins expressed in the vertebrate retina. Eur. J. Neurosci.11:1883–1890. [DOI] [PubMed] [Google Scholar]
- White, T. W., and Paul, D. L. (1999). Genetic diseases and gene knockouts reveal diverse connexin functions.Annu. Rev. Physiol.61:283–310. [DOI] [PubMed] [Google Scholar]
- Wu, G., and Hamill, O. P. (1992). NPPB block of Ca2+-activated Cl- currents in Xenopusoocytes. Eur. J. Physiol.420:227–229. [DOI] [PubMed] [Google Scholar]
- Yamamoto, F., Borgula, G. A., and Steinberg, R. H. (1992). Effects of light and darkness on pH outside rod photoreceptors in the cat retina. Exp. Eye Res.54:685–697. [DOI] [PubMed] [Google Scholar]
- Ye, Z.-C., Wyeth, M. S., Baltan-Tekkok, S., and Ransom, B. R. (2003). Functional hemichannels in astrocytes: A novel mechanism of glutamate release. J. Neurosci.23:3588–3596. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yeager,M., Unger, V., and Falk, M. M. (1998). Synthesis, assembly and structure of gap junction intercellular channels. Curr. Opin. Struct. Biol.8:517–524. [DOI] [PubMed] [Google Scholar]
- Yeh, J. Z., and Narahashi, T. J. (1976). Mechanism of action of quinidine on squid axon membranes. Pharm. Exp. Ther.196:62–70. [PubMed] [Google Scholar]
- Zhang, J.-T., Chen, M., Foote,C. I., and Nicholson,B. J. (1996). Membrane integration of in vitro-translated gap junctional proteins: Co-and post-translational mechanism. Mol. Biol. Cell7:471–482. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang, J.-T., and Nicholson, B. J. (1989). Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its DNA. J. Cell Biol.109:3391–3410. [DOI] [PMC free article] [PubMed] [Google Scholar]