Direct cell-to-cell diffusion of ions and cytosolic molecules is mediated by gap junction (GJ) channels. Each channel is a multimer of connexin (Cx) subunits that forms an intercellular aqueous pore b docking two pre-assembled hemichannels, one from each of two apposed cells.
The first aim of this proposal is to study GJ channel formation as it relates to the events that occur after the establishment of cell-cell contact We utilize our ability to manipulate cells into contact while recording and uniquely combine electrophysiology of GJ formation with fluorescence imaging of Cx distribution in vivo real time. Cxs will be fused with enhanced GFP to allow their visualization. We will examine how the dynamics of Cx redistribution, such as plaque formation is associated with the establishment of electrical coupling. We will examine whether Cxs arrive at the junctional membrane from cytoplasmic stores of adjacent membranes and how membrane fluidity and extracellular Ca2+ affect the association between Cx distribution and GJ formation. We will also study the biophysical properties of de novo GJ channel openings, a process that we propose is a form of gating elicited by hemichannel docking.
The second aim of the proposal is to test the hypothesis that the de novo opening of a GJ channel involves Cx domain(s) that are part of a ~common~ gate that is acted upon by different agents. Based on our studies of voltage and chemical gating of GJ channels formed of different Cxs, activators of this common gate include alkanols, H+ and membrane voltage. These agents close GJ channels completely, and the ~common gate~ may represent the principal means by which coupling is dynamically regulated in cell populations where voltages remain uniform. We will also examine gating by transjunctional voltage (Vi). We demonstrate that the Vi gate only partially closes GJ channels to a residual state whose conductance is Cx specific. We will examine the ionic selectivity of the residual state and will assess the pore size of the residual and fully open states by measuring intercellular diffusion of uncharged dyes.
The third aim of this proposal is to explore how Vi gating combined with the rectifying properties of open and residual states can play a role in regulating electronic transmission between excitable cells. Using heterotypic junctions, with combinations of Cxs that are present in the nervous system, we demonstrate conditions under which some heterotypic junctions exhibit nearly unidirectional transmission. These studies focus on defining the mechanisms that control GJ channel formation and gating which are ultimately necessary for determining the role of intercellular communication in normal and diseased states. Human genetic disorders that have been linked to Cx dysfunction include C-linked CMT disease, visceroatrial heterotaxia and sensorineural deafness.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS036706-03
Application #
6351847
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Stewart, Randall
Project Start
1999-02-01
Project End
2003-01-31
Budget Start
2001-02-01
Budget End
2002-01-31
Support Year
3
Fiscal Year
2001
Total Cost
$187,956
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Neurosciences
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Palacios-Prado, Nicolas; Briggs, Stephen W; Skeberdis, Vytenis A et al. (2010) 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
Garre, Juan M; Retamal, Mauricio A; Cassina, Patricia et al. (2010) FGF-1 induces ATP release from spinal astrocytes in culture and opens pannexin and connexin hemichannels. Proc Natl Acad Sci U S A 107:22659-64
Paulauskas, Nerijus; Pranevicius, Mindaugas; Pranevicius, Henrikas et al. (2009) A stochastic four-state model of contingent gating of gap junction channels containing two ""fast"" gates sensitive to transjunctional voltage. Biophys J 96:3936-48
Sonntag, Stephan; Söhl, Goran; Dobrowolski, Radoslaw et al. (2009) Mouse lens connexin23 (Gje1) does not form functional gap junction channels but causes enhanced ATP release from HeLa cells. Eur J Cell Biol 88:65-77
Palacios-Prado, Nicolas; Sonntag, Stephan; Skeberdis, Vytenis A et al. (2009) Gating, permselectivity and pH-dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina. J Physiol 587:3251-69
Palacios-Prado, Nicolas; Bukauskas, Feliksas F (2009) Heterotypic gap junction channels as voltage-sensitive valves for intercellular signaling. Proc Natl Acad Sci U S A 106:14855-60
Dobrowolski, Radoslaw; Sasse, Philipp; Schrickel, Jan W et al. (2008) The conditional connexin43G138R mouse mutant represents a new model of hereditary oculodentodigital dysplasia in humans. Hum Mol Genet 17:539-54
Rackauskas, Mindaugas; Verselis, Vytas K; Bukauskas, Feliksas F (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:H1729-36
Rackauskas, Mindaugas; Kreuzberg, Maria M; Pranevicius, Mindaugas et al. (2007) Gating properties of heterotypic gap junction channels formed of connexins 40, 43, and 45. Biophys J 92:1952-65
Kreuzberg, Maria M; Willecke, Klaus; Bukauskas, Feliksas F (2006) Connexin-mediated cardiac impulse propagation: connexin 30.2 slows atrioventricular conduction in mouse heart. Trends Cardiovasc Med 16:266-72

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