Abstract
It is now clear that connexin-based, gap junction “hemichannels” in an undocked state are capable of opening and connecting cytoplasm to the extracellular milieu. Varied studies also suggest that such channel activity plays a vital role in diverse cell processes and abnormal hemichannel activity contributes to pathogenesis. To pursue fundamental questions in this area, investigators require methods for studying hemichannel permeability and dynamics that are quantitative, sensitive, versatile, and available to most cellular and molecular laboratories. Here we first provide a theoretical background for this work, including the role of cellular membrane potentials. We then describe in detail our computer-assisted methods for both dye uptake and leakage along with illustrative results from different cell systems. A key feature of our protocol is the inclusion of a mechanical stimulation step. We describe dye uptake, interpreted as connexin dependent, that is shown to be enhanced with reduced extracellular Ca2+, mechanically responsive, inhibited by TPA, inhibited by EL186 antibodies for Cx43 and sustained for more than 15 min following mechanical stimulation. We describe dye leakage that displays these same properties, with estimates of hemichannel numbers per cell being derived from leakage rates. We also describe dye uptake that is shown to be unaffected by a reduction in external Ca2+, insensitive to EL186 antibodies and relatively short-lived following mechanical stimulation; this uptake may occur via pannexin 1 channels expressed in the cells studied here. It is unlikely that cell damage plays a significant role in dye uptake following mechanical stimulation, given compelling results from various control experiments.
Similar content being viewed by others
References
Abudara V, Bechberger J, Freitas-Andrade M, De Bock M, Wang N, Bultynck G, Naus CC, Leybaert L, Giaume C (2014) The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes. Front Cell Neurosci 8:306
Atkinson MM, Sheridan JD (1988) Altered junctional permeability between cells transformed by v-ras, v-mos, or v-src. Am J Physiol 255:C674–C683
Bao L, Locovei S, Dahl G (2004a) Pannexin membrane channels are mechanosensitive conduits for ATP. FEBS Lett 572:65–68
Bao L, Sachs F, Dahl G (2004b) Connexins are mechanosensitive. Am J Physiol Cell Physiol 287:C1389–C1395
Bao X, Lee SC, Reuss L, Altenberg GA (2007) Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC. Proc Natl Acad Sci U S A 104:4919–4924
Baranova A, Ivanov D, Petrash N, Pestova A, Skoblov M, Kelmanson I, Shagin D, Nazarenko S, Geraymovych E, Litvin O et al (2004) The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins. Genomics 83:706–716
Batra N, Burra S, Siller-Jackson AJ, Gu S, Xia X, Weber GF, DeSimone D, Bonewald LF, Lafer EM, Sprague E et al (2012) Mechanical stress-activated integrin alpha5beta1 induces opening of connexin 43 hemichannels. Proc Natl Acad Sci U S A 109:3359–3364
Batra N, Riquelme MA, Burra S, Kar R, Gu S, Jiang JX (2014) Direct regulation of osteocytic connexin 43 hemichannels through AKT kinase activated by mechanical stimulation. J Biol Chem 289:10582–10591
Baumgart T, Hammond AT, Sengupta P, Hess ST, Holowka DA, Baird BA, Webb WW (2007) Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proc Natl Acad Sci U S A 104:3165–3170
Bennett MV, Garre JM, Orellana JA, Bukauskas FF, Nedergaard M, Sáez JC (2012) Connexin and pannexin hemichannels in inflammatory responses of glia and neurons. Brain Res 1487:3–15
Biegon RP, Atkinson MM, Liu TF, Kam EY, Sheridan JD (1987) Permeance of Novikoff hepatoma gap junctions: quantitative video analysis of dye transfer. J Membr Biol 96:225–233
Billinton N, Knight AW (2001) Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal Biochem 291:175–197
Blicher A, Wodzinska K, Fidorra M, Winterhalter M, Heimburg T (2009) The temperature dependence of lipid membrane permeability, its quantized nature, and the influence of anesthetics. Biophys J 96:4581–4591
Bruzzone S, Guida L, Zocchi E, Franco L, De Flora A (2001) Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells. FASEB J 15:10–12
Bruzzone R, Hormuzdi SG, Barbe MT, Herb A, Monyer H (2003) Pannexins, a family of gap junction proteins expressed in brain. Proc Natl Acad Sci U S A 100:13644–13649
Bruzzone R, Barbe MT, Jakob NJ, Monyer H (2005) Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes. J Neurochem 92:1033–1043
Chekeni FB, Elliott MR, Sandilos JK, Walk SF, Kinchen JM, Lazarowski ER, Armstrong AJ, Penuela S, Laird DW, Salvesen GS et al (2010) Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 467:863–867
Cherian PP, Siller-Jackson AJ, Gu S, Wang X, Bonewald LF, Sprague E, Jiang JX (2005) Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin. Mol Biol Cell 16:3100–3106
Contreras JE, Sanchez HA, Eugenin EA, Speidel D, Theis M, Willecke K, Bukauskas FF, Bennett MV, Sáez JC (2002) Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. Proc Natl Acad Sci U S A 99:495–500
Contreras JE, Sáez JC, Bukauskas FF, Bennett MV (2003) Gating and regulation of connexin 43 (Cx43) hemichannels. Proc Natl Acad Sci U S A 100:11388–11393
De Vuyst E, Wang N, Decrock E, De Bock M, Vinken M, Van Moorhem M, Lai C, Culot M, Rogiers V, Cecchelli R et al (2009) Ca(2+) regulation of connexin 43 hemichannels in C6 glioma and glial cells. Cell Calcium 46:176–187
DeVries SH, Schwartz EA (1992) Hemi-gap-junction channels in solitary horizontal cells of the catfish retina. J Physiol 445:201–230
D’Hondt C, Iyyathurai J, Himpens B, Leybaert L, Bultynck G (2014) Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond. Front Physiol 5:348
Dobrowolski R, Willecke K (2009) Connexin-caused genetic diseases and corresponding mouse models. Antioxid Redox Signal 11:283–295
Ebihara L (1996) Xenopus connexin38 forms hemi-gap-junctional channels in the nonjunctional plasma membrane of Xenopus oocytes. Biophys J 71:742–748
Eckert R (2006) Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures. Biophys J 91:565–579
Franco L, Zocchi E, Usai C, Guida L, Bruzzone S, Costa A, De Flora A (2001) Paracrine roles of NAD+ and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts. J Biol Chem 276:21642–21648
Garre JM, Retamal MA, Cassina P, Barbeito L, Bukauskas FF, Sáez JC, Bennett MV, Abudara V (2010) FGF-1 induces ATP release from spinal astrocytes in culture and opens pannexin and connexin hemichannels. Proc Natl Acad Sci U S A 107:22659–22664
Giaume C, Orellana JA, Abudara V, Sáez JC (2012) Connexin-based channels in astrocytes: how to study their properties. Methods Mol Biol 814:283–303
Hansen DB, Ye ZC, Calloe K, Braunstein TH, Hofgaard JP, Ransom BR, Nielsen MS, MacAulay N (2014) Activation, permeability, and inhibition of astrocytic and neuronal large pore (hemi)channels. J Biol Chem 289:26058–26073
Hofer A, Dermietzel R (1998) Visualization and functional blocking of gap junction hemichannels (connexons) with antibodies against external loop domains in astrocytes. Glia 24:141–154
Hur KC, Shim JE, Johnson RG (2003) A potential role for Cx43-hemichannels in staurosporin-induced apoptosis. Cell Commun Adhes 10:271–277
Iglesias R, Locovei S, Roque A, Alberto AP, Dahl G, Spray DC, Scemes E (2008) P2X7 receptor-Pannexin1 complex: pharmacology and signaling. Am J Physiol Cell Physiol 295:C752–C760
John SA, Kondo R, Wang SY, Goldhaber JI, Weiss JN (1999) Connexin-43 hemichannels opened by metabolic inhibition. J Biol Chem 274:236–240
Johnson RG, Meyer RA, Li XR, Preus DM, Tan L, Grunenwald H, Paulson AF, Laird DW, Sheridan JD (2002) Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules. Exp Cell Res 275:67–80
Johnson SA, Stinson BM, Go MS, Carmona LM, Reminick JI, Fang X, Baumgart T (2010) Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles. Biochim Biophys Acta 1798:1427–1435
Johnson RG, Reynhout JK, TenBroek EM, Quade BJ, Yasumura T, Davidson KG, Sheridan JD, Rash JE (2012) Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43. Mol Biol Cell 23:71–86
Kalvelyte A, Imbrasaite A, Bukauskiene A, Verselis VK, Bukauskas FF (2003) Connexins and apoptotic transformation. Biochem Pharmacol 66:1661–1672
Kamkin A, Kiseleva I, Wagner KD, Pylaev A, Leiterer KP, Theres H, Scholz H, Gunther J, Isenberg G (2002) A possible role for atrial fibroblasts in postinfarction bradycardia. Am J Physiol Heart Circ Physiol 282:H842–H849
Kamkin A, Kiseleva I, Wagner KD, Lozinsky I, Gunther J, Scholz H (2003) Mechanically induced potentials in atrial fibroblasts from rat hearts are sensitive to hypoxia/reoxygenation. Pflugers Arch 446:169–174
Kang J, Kang N, Lovatt D, Torres A, Zhao Z, Lin J, Nedergaard M (2008) Connexin 43 hemichannels are permeable to ATP. J Neurosci 28:4702–4711
Klaassen LJ, Sun Z, Steijaert MN, Bolte P, Fahrenfort I, Sjoerdsma T, Klooster J, Claassen Y, Shields CR, Ten Eikelder HM et al (2011) Synaptic transmission from horizontal cells to cones is impaired by loss of connexin hemichannels. PLoS Biol 9:e1001107
Leybaert L, Braet K, Vandamme W, Cabooter L, Martin PE, Evans WH (2003) Connexin channels, connexin mimetic peptides and ATP release. Cell Commun Adhes 10:251–257
Li H, Liu TF, Lazrak A, Peracchia C, Goldberg GS, Lampe PD, Johnson RG (1996) Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells. J Cell Biol 134:1019–1030
Liu TF, Li HY, Atkinson MM, Johnson RG (1995) Intracellular lucifer yellow leakage from Novikoff cells in the presence of ATP or low extracellular Ca2+: evidence for hemi-gap junction channels. Methods Find Exp Clin Pharmacol 17:23–28
Locovei S, Bao L, Dahl G (2006) Pannexin 1 in erythrocytes: function without a gap. Proc Natl Acad Sci U S A 103:7655–7659
Meyer RA, Laird DW, Revel JP, Johnson RG (1992) Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies. J Cell Biol 119:179–189
Orellana JA, Hernandez DE, Ezan P, Velarde V, Bennett MV, Giaume C, Sáez JC (2010) Hypoxia in high glucose followed by reoxygenation in normal glucose reduces the viability of cortical astrocytes through increased permeability of connexin 43 hemichannels. Glia 58:329–343
Orellana JA, Diaz E, Schalper KA, Vargas AA, Bennett MV, Sáez JC (2011a) Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species. Biochem Biophys Res Commun 409:603–609
Orellana JA, Froger N, Ezan P, Jiang JX, Bennett MV, Naus CC, Giaume C, Sáez JC (2011b) ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels. J Neurochem 118:826–840
Orellana JA, Shoji KF, Abudara V, Ezan P, Amigou E, Sáez PJ, Jiang JX, Naus CC, Sáez JC, Giaume C (2011c) Amyloid beta-induced death in neurons involves glial and neuronal hemichannels. J Neurosci 31:4962–4977
Orellana JA, Sáez PJ, Cortes-Campos C, Elizondo RJ, Shoji KF, Contreras-Duarte S, Figueroa V, Velarde V, Jiang JX, Nualart F et al (2012) Glucose increases intracellular free Ca(2+) in tanycytes via ATP released through connexin 43 hemichannels. Glia 60:53–68
Palacios-Prado N, Bukauskas FF (2009) Heterotypic gap junction channels as voltage-sensitive valves for intercellular signaling. Proc Natl Acad Sci U S A 106:14855–14860
Panchin YV (2005) Evolution of gap junction proteins—the pannexin alternative. J Exp Biol 208:1415–1419
Paul DL, Ebihara L, Takemoto LJ, Swenson KI, Goodenough DA (1991) Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell Biol 115:1077–1089
Penuela S, Bhalla R, Gong XQ, Cowan KN, Celetti SJ, Cowan BJ, Bai D, Shao Q, Laird DW (2007) Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins. J Cell Sci 120:3772–3783
Penuela S, Gehi R, Laird DW (2013) The biochemistry and function of pannexin channels. Biochim Biophys Acta 1828:15–22
Phelan P, Bacon JP, Davies JA, Stebbings LA, Todman MG, Avery L, Baines RA, Barnes TM, Ford C, Hekimi S et al (1998) Innexins: a family of invertebrate gap-junction proteins. Trends Genet 14:348–349
Quist AP, Rhee SK, Lin H, Lal R (2000) Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol 148:1063–1074
Rackauskas M, Verselis VK, Bukauskas FF (2007) Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45. Am J Physiol Heart Circ Physiol 293:H1729–H1736
Riquelme MA, Kar R, Gu S, Jiang JX (2013) Antibodies targeting extracellular domain of connexins for studies of hemichannels. Neuropharmacology 75:525–532
Sáez JC, Leybaert L (2014) Hunting for connexin hemichannels. FEBS Lett 588:1205–1211
Sáez JC, Contreras JE, Bukauskas FF, Retamal MA, Bennett MV (2003) Gap junction hemichannels in astrocytes of the CNS. Acta Physiol Scand 179:9–22
Sáez JC, Retamal MA, Basilio D, Bukauskas FF, Bennett MV (2005) Connexin-based gap junction hemichannels: gating mechanisms. Biochim Biophys Acta 1711:215–224
Sáez JC, Schalper KA, Retamal MA, Orellana JA, Shoji KF, Bennett MV (2010) Cell membrane permeabilization via connexin hemichannels in living and dying cells. Exp Cell Res 316:2377–2389
Sahu G, Sukumaran S, Bera AK (2014) Pannexins form gap junctions with electrophysiological and pharmacological properties distinct from connexins. Sci Rep 4:4955
Sangaletti R, Dahl G, Bianchi L (2014) Mechanosensitive unpaired innexin channels in C. elegans touch neurons. Am J Physiol Cell Physiol 307:C966–C977
Scemes E (2011) Nature of plasmalemmal functional “hemichannels”. Biochim Biophys Acta 1818:1880–1883
Schalper KA, Palacios-Prado N, Retamal MA, Shoji KF, Martinez AD, Sáez JC (2008) Connexin hemichannel composition determines the FGF-1-induced membrane permeability and free [Ca2+]i responses. Mol Biol Cell 19:3501–3513
Siebert AP, Ma Z, Grevet JD, Demuro A, Parker I, Foskett JK (2013) Structural and functional similarities of calcium homeostasis modulator 1 (CALHM1) ion channel with connexins, pannexins, and innexins. J Biol Chem 288:6140–6153
Sipos A, Vargas SL, Toma I, Hanner F, Willecke K, Peti-Peterdi J (2009) Connexin 30 deficiency impairs renal tubular ATP release and pressure natriuresis. J Am Soc Nephrol 20:1724–1732
Sosinsky GE, Boassa D, Dermietzel R, Duffy HS, Laird DW, MacVicar B, Naus CC, Penuela S, Scemes E, Spray DC et al (2011) Pannexin channels are not gap junction hemichannels. Channels (Austin) 5:193–197
Spitzer N, Sammons GS, Price EM (2011) Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies. J Neurosci Methods 197:48–55
Spray DC, Ye ZC, Ransom BR (2006) Functional connexin “hemichannels”: a critical appraisal. Glia 54:758–773
Starich TA, Lee RY, Panzarella C, Avery L, Shaw JE (1996) eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling. J Cell Biol 134:537–548
Stout CE, Costantin JL, Naus CC, Charles AC (2002) Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J Biol Chem 277:10482–10488
Swenson ES, Price JG, Brazelton T, Krause DS (2007) Limitations of green fluorescent protein as a cell lineage marker. Stem Cells 25:2593–2600
TenBroek EM, Lampe PD, Solan JL, Reynhout JK, Johnson RG (2001) Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP. J Cell Biol 155:1307–1318
Trexler EB, Bennett MV, Bargiello TA, Verselis VK (1996) Voltage gating and permeation in a gap junction hemichannel. Proc Natl Acad Sci U S A 93:5836–5841
Trexler EB, Bukauskas FF, Bennett MV, Bargiello TA, Verselis VK (1999) Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation. J Gen Physiol 113:721–742
Valiunas V (2002) Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells. J Gen Physiol 119:147–164
Verselis V, White RL, Spray DC, Bennett MV (1986) Gap junctional conductance and permeability are linearly related. Science 234:461–464
Wang N, De Bock M, Antoons G, Gadicherla AK, Bol M, Decrock E, Evans WH, Sipido KR, Bukauskas FF, Leybaert L (2012) Connexin mimetic peptides inhibit Cx43 hemichannel opening triggered by voltage and intracellular Ca2+ elevation. Basic Res Cardiol 107:304
Ye ZC, Wyeth MS, Baltan-Tekkok S, Ransom BR (2003) Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J Neurosci 23:3588–3596
Yeager M, Harris AL (2007) Gap junction channel structure in the early 21st century: facts and fantasies. Curr Opin Cell Biol 19:521–528
Acknowledgments
We gratefully acknowledge the generosity of Drs. Alan Lau and Bonnie Cramer, Klaus Willecke and Claudia Elfgang, as well as Christian Naus, for providing cell lines used in some of these studies. We also thank Drs. Jim Nagy and Gerhard Dahl for providing pannexin 1 antibodies. This work was supported by NIH Grant GM-46277 to RGJ and JDS. RGJ initiated these studies, developed the basic protocols for both mechanical stimulation and dye uptake, performed some of the early experiments, and supervised the work with the connexin antibodies. JDS developed and modeled the theoretical background for this work, including the explicit inclusion of membrane potential effects on uptake and leakage, and created the basic macros and protocols for imaging cells and analyzing fluorescence intensities. He also supervised the work on fluorescence intensities. The work described in Online Resource 7 was carried out by BAC in the JCS laboratory, with RGJ’s involvement in the initial studies. LTF performed dye leakage experiments that informed similar experiments reported here. DWL developed the peptide-specific antibodies for Cx43 used here. All other co-authors were students or technicians in the Johnson-Sheridan lab who performed experiments that led to the development of the uptake/leakage methods or provided the findings described here. RGJ and JDS also wrote this manuscript.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Johnson, R.G., Le, H.C., Evenson, K. et al. Connexin Hemichannels: Methods for Dye Uptake and Leakage. J Membrane Biol 249, 713–741 (2016). https://doi.org/10.1007/s00232-016-9925-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00232-016-9925-y