Abstract
Gap junctions are clusters of transmembrane channels allowing a passive diffusion of ions and small molecules between adjacent cells. Connexin43, the main channel-forming protein expressed in ventricular myocytes, can associate with zonula occludens-1, a scaffolding protein linked to the actin cytoskeleton and to signal transduction molecules. The possible influence of Rho GTPases, major regulators of cellular junctions and of the actin cytoskeleton, in the modulation of gap junctional intercellular communication (GJIC) was examined. The activation of RhoA by cytoxic necrotizing factor 1 markedly enhanced GJIC, whereas its specific inhibition by the Clostridium botulinum C3 exoenzyme significantly reduced it. RhoA activity affects GJIC without major cellular redistribution of junctional plaques or changes in the Cx43 phosphorylation pattern. As these GTPases frequently act via the cortical cytoskeleton, the importance of F-actin in the modulation of GJIC was investigated by means of agents interfering with actin polymerization. Cytoskeleton stabilization by phalloidin slowed down the kinetics of channel rundown in the absence of ATP, whereas its disruption by cytochalasin D rapidly and markedly reduced GJIC despite ATP presence. Cytoskeleton stabilization by phalloidin markedly reduced the consequences of RhoA activation or inactivation. This mechanism appears to be the first described capable to both up- or down-regulate GJIC through RhoA activation or, conversely, inhibition. The inhibition of Rho downstream kinase effectors had no effect on GJIC. The present results provide further insight into the gating and regulation of junctional channels and identify a new downstream target for the small G-protein RhoA.
Highlights
Nel results from the docking of two half-channels, or connexons, formed by the oligomerization of six protein subunits (connexins (Cxs))2 around an aqueous pore
The first hypothesis to explain this ability of GTP to preserve gap junctional intercellular communication (GJIC) would be that GTP can serve as co-substrate to the up to now unidentified protein kinase (PK) that would counterbalance phosphatase 1 (PP1) activity
Because (i) actin filament depolymerization by cytochalasin D elicited a junctional current rundown despite the presence of ATP whereas (ii) actin filament stabilization by phalloidin slowed down the loss of junctional channel activity in ATP-deprived conditions and markedly reduced the effects of RhoA activation or inactivation, RhoA is likely to control the degree of intercellular coupling via its pivotal role in regulating the actin cytoskeleton
Summary
Connexin; ZO-1, zonula occludens-1; CNF, cytoxic necrotizing factor; GJIC, gap junctional intercellular communication; PP, protein phosphatase; PK, protein kinase; PKN, protein kinase novel; GTPase, guanosine triphosphatase; GJ, gap junction; TRITC, tetramethylrhodamine isothiocyanate; ROCK, Rho-associated coiled coil-containing kinase; CRIK, citron kinase; Gj, conductance; AMP-PNP, adenosine 5Ј-(,␥-imino)triphosphate; FRAP, fluorescence recovery after photobleaching. Many aspects of connexin function, for example intracellular cellular transport, junctional plaque assembly and stability, and channel conductivity, are finely tuned and likely involve proteins that bind to the cytoplasmic domains of connexins. Cardiac-specific inhibition and activation of RhoA signaling are both known to result in alterations of cardiac rhythm and conduction, processes where GJIC is of fundamental importance
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