Skip to main content
Springer Nature Link
Log in

Neurons and β-Cells of the Pancreas Express Connexin36, Forming Gap Junction Channels that Exhibit Strong Cationic Selectivity

  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and β-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (P γ), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye transfer at levels not resolvable using whole-field excitation solely. We demonstrate that P γ of Cx36 for cationic dyes (EAM-1+ and EAM-2+) is ~10-fold higher than that for an anionic dye of the same net charge and similar molecular mass, Alexa fluor-350 (AFl-350−). In addition, P γ for Lucifer yellow (LY2−) is approximately fourfold smaller than that for AFl-350−, which suggests that the higher negativity of LY2− significantly reduces permeability. The P γ of Cx36 for AFl-350 is approximately 358, 138, 23 and four times smaller than the P γs of Cx43, Cx40, Cx45, and Cx57, respectively. In contrast, it is 6.5-fold higher than the P γ of mCx30.2, which exhibits a smaller single-channel conductance. Thus, Cx36 GJs are highly cation-selective and should exhibit relatively low permeability to numerous vital negatively charged metabolites and high permeability to K+, a major charge carrier in cell–cell communication.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
€32.70 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Finland)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Bednarczyk D, Mash EA, Aavula BR, Wright SH (2000) NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules. Pflugers Arch 440:184–192

    PubMed  CAS  Google Scholar 

  • Bedner P, Niessen H, Odermatt B, Kretz M, Willecke K, Harz H (2006) Selective permeability of different connexin channels to the second messenger cyclic AMP. J Biol Chem 28:6673–6681

    Article  Google Scholar 

  • Bennett MV, Verselis VK (1992) Biophysics of gap junctions. Semin Cell Biol 3:29–47

    Article  PubMed  CAS  Google Scholar 

  • Bukauskas FF (2001) Inducing de novo formation of gap junction channels. Method Mol Biol 154:379–393

    CAS  Google Scholar 

  • Bukauskas FF, Peracchia C (1997) Two distinct gating mechanisms in gap junction channels: CO2-sensitive and voltage-sensitive. Biophys J 72:2137–2142

    Article  PubMed  CAS  Google Scholar 

  • Bukauskas FF, Verselis VK (2004) Gap junction channel gating. Biochim Biophys Acta 1662:42–60

    Article  PubMed  CAS  Google Scholar 

  • Bukauskas FF, Weingart R (1994) Voltage-dependent gating of single gap junction channels in an insect cell line. Biophys J 67:613–625

    Article  PubMed  CAS  Google Scholar 

  • Bukauskas FF, Bukauskiene A, Verselis VK (2002a) Conductance and permeability of the residual state of connexin43 gap junction channels. J Gen Physiol 119:171–186

    Article  PubMed  CAS  Google Scholar 

  • Bukauskas FF, Bukauskiene A, Verselis VK, Bennett MVL (2002b) Coupling asymmetry of heterotypic connexin 45/connexin 43-EGFP gap junctions: properties of fast and slow gating mechanisms. Proc Natl Acad Sci USA 99:7113–7118

    Article  PubMed  CAS  Google Scholar 

  • Campbell RE, Ducret E, Porteous R, Liu X, Herde MK, Wellerhaus K, Sonntag S, Willecke K, Herbison AE (2011) Gap junctions between neuronal inputs but not gonadotropin-releasing hormone neurons control estrous cycles in the mouse. Endocrinology 152:2290–2301

    Article  PubMed  CAS  Google Scholar 

  • Charpantier E, Cancela J, Meda P (2007) Beta cells preferentially exchange cationic molecules via connexin 36 gap junction channels. Diabetologia 50:2332–2341

    Article  PubMed  CAS  Google Scholar 

  • Condorelli DF, Parenti R, Spinella F, Salinaro AT, Belluardo N, Cardile V, Cicirata F (1998) Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. Eur J Neurosci 10(3):1202–1208

    Article  PubMed  CAS  Google Scholar 

  • Dobrenis K, Chang H, Pina-Benabou MH, Woodroffe A, Lee SC, Rozental R, Spray DC, Scemes E (2005) Human and mouse microglia express connexin36, and functional gap junctions are formed between rodent microglia and neurons. J Neurosci Res 82:306–315

    Article  PubMed  CAS  Google Scholar 

  • Eckert R (2006) Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures. Biophys J 91:565–579

    Article  PubMed  CAS  Google Scholar 

  • Ek-Vitorin JF, Burt JM (2005) Quantification of gap junction selectivity. Am J Physiol Cell Physiol 289:C1535–C1546

    Article  PubMed  CAS  Google Scholar 

  • Ek-Vitorin JF, King TJ, Heyman NS, Lampe PD, Burt JM (2006) Selectivity of connexin 43 channels is regulated through protein kinase C–dependent phosphorylation. Circ Res 98:1498–1505

    Article  PubMed  CAS  Google Scholar 

  • Goldberg GS, Valiunas V, Brink PR (2004) Selective permeability of gap junction channels. Biochim Biophys Acta 1662:96–101

    Article  PubMed  CAS  Google Scholar 

  • Harris AL (2001) Emerging issues of connexin channels: biophysics fills the gap. Q Rev Biophys 34:325–427

    PubMed  CAS  Google Scholar 

  • Harris AL (2007) Connexin channel permeability to cytoplasmic molecules. Prog Biophys Mol Biol 94:120–143

    Article  PubMed  CAS  Google Scholar 

  • Hernandez VH, Bortolozzi M, Pertegato V, Beltramello M, Giarin M, Zaccolo M, Pantano S, Mammano F (2007) Unitary permeability of gap junction channels to second messengers measured by FRET microscopy. Nat Methods 4:353–358

    PubMed  CAS  Google Scholar 

  • Heyman NS, Burt JM (2008) Hindered diffusion through an aqueous pore describes invariant dye selectivity of Cx43 junctions. Biophys J 94:840–854

    Article  PubMed  CAS  Google Scholar 

  • Heyman NS, Kurjiaka DT, Ek-Vitorin JF, Burt JM (2009) Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43. Am J Physiol Heart Circ Physiol 297:H450–H459

    Article  PubMed  CAS  Google Scholar 

  • Hille B (2001) Ionic channels of excitable membranes. Sinauer Associates, Sunderland

    Google Scholar 

  • Jain R, Lammert E (2009) Cell–cell interactions in the endocrine pancreas. Diabetes Obes Metab 11:159–167

    Article  PubMed  CAS  Google Scholar 

  • Kanaporis G, Brink PR, Valiunas V (2011) Gap junction permeability: selectivity for anionic and cationic probes. Am J Physiol Cell Physiol 300:C600–C609

    Article  PubMed  CAS  Google Scholar 

  • Kreuzberg MM, Sohl G, Kim J, Verselis VK, Willecke K, Bukauskas FF (2005) Functional properties of mouse connexin30.2 expressed in the conduction system of the heart. Circ Res 96:1169–1177

    Article  PubMed  CAS  Google Scholar 

  • Nitsche JM, Chang H, Weber PA, Nicholson BJ (2004) A transient diffusion model yields unitary gap junctional permeabilities from images of cell-to-cell fluorescent dye transfer between Xenopus oocytes. Biophys J 86:2058–2077

    Article  PubMed  CAS  Google Scholar 

  • Palacios-Prado N, Bukauskas FF (2009) Heterotypic gap junction channels as voltage-sensitive valves for intercellular signaling. Proc Natl Acad Sci USA 106:14855–14860

    Article  PubMed  CAS  Google Scholar 

  • Palacios-Prado N, Bukauskas FF (2012) Modulation of metabolic communication through gap junction channels by transjunctional voltage; synergistic and antagonistic effects of gating and ionophoresis. Biochim Biophys Acta 1818:1884–1894

    Article  PubMed  CAS  Google Scholar 

  • Palacios-Prado N, Sonntag S, Skeberdis VA, Willecke K, Bukauskas FF (2009) Gating, permselectivity and pH-dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina. J Physiol 587:3251–3269

    Article  PubMed  CAS  Google Scholar 

  • Palacios-Prado N, Briggs SW, Skeberdis VA, Pranevicius M, Bennett MV, Bukauskas FF (2010) pH-dependent modulation of voltage gating in connexin45 homotypic and connexin45/connexin43 heterotypic gap junctions. Proc Natl Acad Sci USA 107:9897–9902

    Article  PubMed  CAS  Google Scholar 

  • Pan F, Paul DL, Bloomfield SA, Völgyi B (2010) Connexin36 is required for gap junctional coupling of most ganglion cell subtypes in the mouse retina. J Comp Neurol 518:911–927

    Article  PubMed  Google Scholar 

  • Peracchia C (2004) Chemical gating of gap junction channels; roles of calcium, pH and calmodulin. Biochim Biophys Acta 1662:61–80

    Article  PubMed  CAS  Google Scholar 

  • Pereda AE, Bell TD, Faber DS (1995) Retrograde synaptic communication via gap junctions coupling auditory afferents to the Mauthner cell. J Neurosci 15:5943–5955

    PubMed  CAS  Google Scholar 

  • Pereda A, O’Brien J, Nagy JI, Bukauskas F, Davidson KG, Kamasawa N, Yasumura T, Rash JE (2003) Connexin35 mediates electrical transmission at mixed synapses on Mauthner cells. J Neurosci 23:7489–7503

    PubMed  CAS  Google Scholar 

  • Ponsioen B, van Zeijl L, Moolenaar WH, Jalink K (2007) Direct measurement of cyclic AMP diffusion and signaling through connexin43 gap junctional channels. Exp Cell Res 313:415–423

    Article  PubMed  CAS  Google Scholar 

  • Qu Y, Dahl G (2002) Function of the voltage gate of gap junction channels: selective exclusion of molecules. Proc Natl Acad Sci USA 99:697–702

    Article  PubMed  CAS  Google Scholar 

  • Rackauskas M, Kreuzberg MM, Pranevicius M, Willecke K, Verselis VK, Bukauskas FF (2007a) Gating properties of heterotypic gap junction channels formed of connexins 40, 43 and 45. Biophys J 92:1952–1965

    Article  PubMed  CAS  Google Scholar 

  • Rackauskas M, Verselis VK, Bukauskas FF (2007b) 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(3):H1729–H1736

    Article  PubMed  CAS  Google Scholar 

  • Serre-Beinier V, Le Gurun S, Belluardo N, Trovato-Salinaro A, Charollais A, Haefliger JA, Condorelli DF, Meda P (2000) Cx36 preferentially connects beta-cells within pancreatic islets. Diabetes 49(5):727–734

    Article  PubMed  CAS  Google Scholar 

  • Srinivas M, Rozental R, Kojima T, Dermietzel R, Mehler M, Condorelli DF, Kessler JA, Spray DC (1999) Functional properties of channels formed by the neuronal gap junction protein connexin36. J Neurosci 19:9848–9855

    PubMed  CAS  Google Scholar 

  • Teubner B, Degen J, Sohl G, Guldenagel M, Bukauskas FF, Trexler EB, Verselis VK, De Zeeuw CI, Lee CG, Kozak CA, Petrasch-Parwez E, Dermietzel R, Willecke K (2000) Functional expression of the murine connexin36 gene coding for a neuron-specific gap junctional protein. J Membr Biol 176:249–262

    Article  PubMed  CAS  Google Scholar 

  • Trexler EB, Bennett MV, Bargiello TA, Verselis VK (1996) Voltage gating and permeation in a gap junction hemichannel. Proc Natl Acad Sci USA 93:5836–5841

    Article  PubMed  CAS  Google Scholar 

  • Trexler EB, Bukauskas FF, Bennett MVL, 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

    Article  PubMed  CAS  Google Scholar 

  • Trexler EB, Bukauskas FF, Kronengold J, Bargiello TA, Verselis VK (2000) The first extracellular loop domain is a major determinant of charge selectivity in connexin46 channels. Biophys J 79:3036–3051

    Article  PubMed  CAS  Google Scholar 

  • Veenstra RD, Wang HZ, Beyer EC, Brink PR (1994a) Selective dye and ionic permeability of gap junction channels formed by connexin45. Circ Res 75:483–490

    Article  PubMed  CAS  Google Scholar 

  • Veenstra RD, Wang HZ, Beyer EC, Ramanan SV, Brink PR (1994b) Connexin37 forms high conductance gap junction channels with subconductance state activity and selective dye and ionic permeabilities. Biophys J 66:1915–1928

    Article  PubMed  CAS  Google Scholar 

  • Veenstra RD, Wang HZ, Beblo DA, Chilton MG, Harris AL, Beyer EC, Brink PR (1995) Selectivity of connexin-specific gap junctions does not correlate with channel conductance. Circ Res 77:1156–1165

    Article  PubMed  CAS  Google Scholar 

  • Verselis VK, Veenstra RD (2000) Gap junction channels. Permeability and voltage gating. In: Hertzberg E (ed) Advances in molecular and cell biology, vol 30. JAI Press, Greenwich, pp 129–192

    Google Scholar 

  • Verselis V, White RL, Spray DC, Bennett MV (1986) Gap junctional conductance and permeability are linearly related. Science 234:461–464

    Article  PubMed  CAS  Google Scholar 

  • Verselis VK, Trexler EB, Bukauskas FF (2000) Connexin hemichannels and cell–cell channels: comparison of properties. Braz J Med Biol Res 33:379–389

    Article  PubMed  CAS  Google Scholar 

  • Weber PA, Chang H, Spaeth KE, Nitsche JM, Nicholson BJ (2004) The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities. Biophys J 87:958–973

    Article  PubMed  CAS  Google Scholar 

  • Wilders R, Jongsma HJ (1992) Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels. Biophys J 63:942–953

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. Garry E. Kiefer, chief executive officer of Macrocyclics (Dallas, TX), for the kind supply of EAM-1 and EAM-2 dyes; Dr. Klaus Willecke for kindly providing the constructs of Cx36; and PhD student Nerijus Paulauskas and Dr. Angele Bukauskiene for excellent technical assistance. This work was supported by National Institutes of Health grants HL084464 and NS072238 (to F. F. B.).

Author information

Authors and Affiliations

  1. Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, NY, USA

    Feliksas F. Bukauskas

Authors
  1. Feliksas F. Bukauskas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Feliksas F. Bukauskas.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bukauskas, F.F. Neurons and β-Cells of the Pancreas Express Connexin36, Forming Gap Junction Channels that Exhibit Strong Cationic Selectivity. J Membrane Biol 245, 243–253 (2012). https://doi.org/10.1007/s00232-012-9445-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00232-012-9445-3

Keywords

Search

Navigation