De-novo formation, gating and regulation of gap junction channels and hemichannels.

Our major goals are to study de-novo formation, gating and regulation of gap junction (GJ) channels and unapposed/nonjunctional hemichannels formed by connexin (Cx) proteins. GJ channels mediate direct cell-cell exchange of cytosolic ions and molecules. By combining electrophysiological, imaging and computational modeling methods, we examine electrical cell-cell coupling and metabolic communication under normal conditions and changes of intracellular pH, [Ca2+]i, [Mg2+]i and other reagents in living cells that express different types of wild type Cxs, their mutants and Cxs fused with color variants of green fluorescent protein (Cx-GFP). We demonstrated that in each hemichannel of the GJ channel there are two distinct types of voltage sensitive gating mechanisms, fast and slow/loop, and that the fast gate can serve as a selective filter that preserves electrical cell-cell signaling but restricts metabolic communication and chemical signaling. We developed a stochastic multi-state model describing voltage-gating of homotypic and heterotypic GJ channels combined with methods of global optimization for automated characterization of fast and slow gates from experimental measurements of voltage gating. We propose that clustering of GJ channels into junctional plaques (JPs) is central to their ability to function. We reported that depending on Cx isoform, only ~0.003-0.15 of GJ channels clustered in JPs are functional and this fraction can be significantly modulated by pHi, [Ca2+]i, [Mg2+]i, arachidonic acid, long chain alkanols, albumin and other factors. Furthermore, we demonstrated that heterotypic junctions can exhibit nearly unidirectional electrical signaling and may function as rectifying electrical synapses and that the transjunctional flux of metabolites is affected by ionophoresis and voltage-sensitive gating, which can synergistically or antagonistically affect metabolic communication. Furthermore we study the role of Cxs in the spread of apoptosis and Cx mutants related to deafness, oculodentodigital dysplasia (ODDD), X-linked Charcot-Marie-Tooth disease, cardiac arrhythmia and other hereditary diseases.

Illustration of the function of fast and slow gating mechanisms in fibroblast cell pair expressing Cx43 during uncoupling by CO2 and washout (A), and the schematic of GJ channel with the fast gate (orange arrow) closing the pore partially and the slow gate (blue arrow) closing the pore fully (For more details see Bukauskas and Peracchia, 1997, and Paulauskas et al., 2012).

Publications reflecting major directions of our studies

  • Palacios-Prado, N., Chapuis, S, Panjkovich, A., Fregeac, J., Nagy, J.I. and F.F. Bukauskas. Molecular determinants of magnesium-dependent synaptic plasticity at electrical synapses formed by connexin36. Nature Communications, 2014 Aug 19;5:4667. PMCID: PMC4142521
  • Bukauskas FF. Cardiac Electrophysiology: From Cell to Bedside. 6-th ed. Zipes DP, Jalife J, editors. Philadelphia: ELSEVIER, SAUNDERS; 2014. Chapter 8, Molecular organization, gating and function of gap junction channels. p.85-94.
  • Majoul IV, Gao L, Betzig E, Onichtchouk D, Butkevich E, Kozlov Y, Bukauskas FF, Bennett MV, Lippincott-Schwartz J, Duden R. Fast structural responses of gap junction membrane domains to AB5 toxins. Proc Natl Acad Sci U S A. 2013; 110:E4125-33. PMCID: PMC3816413
  • Lübkemeier I., Andrié R., Lickfett L., Bosen F., Stöckigt F., Dobrowolski R., Draffehn A.M., Fregeac, Schultze J.L., Bukauskas F.F., Schrickel J.W., Willecke K.. The Connexin40A96S mutation from a patient with atrial fibrillation causes decreased atrial conduction velocities and sustained episodes of induced atrial fibrillation in mice. J Mol Cell Cardiol. 2013, 65:19-32. Link
  • Rash J.E., Curti S., Davidson K.G., Kamasawa N., Srikant Nannapaneni, Palacios-Prado N., Flores C.E., Yasumura T., O’Brien J., Lynn B.D., Bukauskas F.F., Nagy J.I., Pereda A.E. Molecular and functional asymmetry at a vertebrate electrical synapse. Neuron. 79:957-69 (2013). Link
  • Lübkemeier I., Requardt R.P., Lin X., Sasse P., Andrié R., Schrickel J.W., Chkourko H., Bukauskas F.F., Kim J.S., Frank M., Malan D., Zhang J., Wirth A., Dobrowolski R., Mohler P.J., Offermanns S., Fleischmann B.K., Delmar M., Willecke K. Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels. Basic Res Cardiol. 108:348 (2013). PMID: 23558439
  • Marandykina A., Palacios-Prado N., Rimkute L., Skeberdis V.A., Bukauskas F.F. Regulation of connexin36 gap junction channels by n-alkanols and arachidonic acid. J Physiol. 591:2087-101 (2013). PMID: 23420660
  • Palacios-Prado N., Hoge G., Marandykina A., Rimkute L., Chapuis S., Paulauskas N., Skeberdis V.A., O'Brien J., Pereda A.E., Bennett M.V., Bukauskas F.F. Intracellular magnesium-dependent modulation of gap junction channels formed by neuronal connexin36. J Neurosci. 33:4741-53 (2013). PMID: 23486946
  • Wang N, De Vuyst E, Ponsaerts R, Boengler K, Palacios-Prado N, Wauman J, Lai CP, De Bock M, Decrock E, Bol M, Vinken M, Rogiers V, Tavernier J, Evans WH, Naus CC, Bukauskas FF, Sipido KR, Heusch G, Schulz R, Bultynck G, Leybaert L. Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury. Basic Res Cardiol. 2013 Jan; 108(1):309. Epub 2012 Nov 8. PMID: 23184389
  • Wang N, De Bock M, Antoons G, Gadicherla AK, Bol M, Decrock E, Evans WH, Sipido KR, Bukauskas FF, Leybaert L. Connexin mimetic peptides inhibit Cx43 hemichannel opening triggered by voltage and intracellular Ca2+ elevation. Basic Res Cardiol. 107(6):304. Epub 2012 Oct 21. PMID: 23095853
  • Bennett MV, Garré JM, Orellana JA, Bukauskas FF, Nedergaard M, Sáez JC. Connexin and pannexin hemichannels in inflammatory responses of glia and neurons. Brain Res. 1487:3-15 (2012).PMID: 22975435
  • Wang N, De Bock M, Decrock E, Bol M, Gadicherla A, Vinken M, Rogiers V, Bukauskas FF, Bultynck G, Leybaert L. Paracrine signaling through plasma membrane hemichannels. Biochim Biophys Acta. 1828:35-50 (2013).PMID: 22796188
  • Eugenin EA, Basilio D, Sáez JC, Orellana JA, Raine CS, Bukauskas F, Bennett MV, Berman JW. The role of gap junction channels during physiologic and pathologic conditions of the human central nervous system. J Neuroimmune Pharmacol. 7:499-518 (2012).PMID: 22438035
  • Bukauskas, F.F. Neurons and ß-cells of pancreas express connexin36 forming gap junction channels exhibiting strong cationic selectivity. J. Membrane Biology. 245:243-53, 2012. PMID: 22752717
  • Paulauskas N, Pranevicius H, Mockus J, Bukauskas FF. A stochastic 16-state model of voltage-gating of gap junction channels enclosing fast and slow gates. Biophysical J. 102:2471-80, 2012.
  • Bukauskas FF. Neurons and ß-cells of pancreas express connexin36 forming gap junction channels exhibiting strong cationic selectivity. J. Membrane Biology. 245:243-53, 2012.
  • Palacios-Prado N, Bukauskas FF. Modulation of metabolic communication through gap junction channels by transjunctional voltage; synergistic and antagonistic effects of gating and ionophoresis. Bioch. et Biophysica Acta. 1818:1884-94, 2012.
  • Skeberdis VA, Rimkute, L, Skeberdyte A, Paulauskas N, Bukauskas FF. pH-dependent modulation of connexin-based gap junctional uncouplers. J. Physiology (London). 15:3495-506, 2011.
  • Palacios-Prado N, Briggs SW, Skeberdis VA, Pranevicius M, Bennett MVL, Bukauskas FF. pH-dependent modulation of voltage gating in connexin45 homotypic and connexin45/connexin43 heterotypic gap junctions. Proc Natl Acad Sci U S A. 107:9897-902, 2010.
  • Palacios-Prado N, Bukauskas FF. Heterotypic Gap Junction Channels as Voltage-Sensitive Valves for Intercellular Signalling. Proc Natl Acad Sci U S A. 106:14855-60. 2009.
  • Paulauskas N, Pranevicius M, Pranevicius H, Bukauskas FF. A stochastic four-state model of contingent gating of gap junction channels containing two 'fast' gates sensitive to transjunctional voltage. Biophysical J. 96:3936-48, 2009.
  • Palacios-Prado N, Sonntag S, Skeberdis VA, Willecke K, Bukauskas FF. Gating, permselectivity and pH-dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina. J. Physiology (London). 587:3251-69, 2009.
  • Rackauskas M, Verselis VK, Bukauskas F. Permeability of homotypic and heterotypic gap junction channels formed of connexins, mCx30.2, Cx40, Cx43 and Cx45. Am. J. Physiol., Heart Circ. Physiol. H1729-36, 2007.
  • Bukauskas FF, Kreuzberg MM, Rackauskas M, Bukauskiene A, Bennett MVL, Verselis VK, Willecke K. Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels; implications for atrioventricular conduction in the heart. Proc Natl Acad Sci U S A. 103:9726-31, 2006.
  • Kreuzberg MM, Willecke K, Bukauskas F. Connexin mediated cardiac impulse propagation: Connexin 30.2 slows atrioventricular conduction in mouse heart. Review, Trends in Cardiovascular Medicine. 16:266-272, 2006.
  • Bukauskas FF, Verselis VK. Gap junction channel gating. Review, Bioch. et Biophysica Acta, 1662:42-60, 2004.