Abstract
Gap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells. Their function is critical for numerous cellular interactions, providing exchange of metabolites, signaling molecules, and ionic currents. GJ channels are composed of Connexin (Cx) hexamers paired across extracellular space and typically form large rafts of clustered channels, called plaques, at cell appositions. Cxs together with molecules that interact with GJ channels make up a supramolecular structure known as the GJ Nexus. While the stability of connexin localization in GJ plaques has been studied, mobility of other Nexus components has yet to be addressed. Colocalization analysis of several nexus components and other membrane proteins reveal that certain molecules are excluded from the GJ plaque (Aquaporin 4, EAAT2b), while others are quite penetrant (lipophilic molecules, Cx30, ZO-1, Occludin). Fluorescence recovery after photobleaching of tagged Nexus-associated proteins showed that mobility in plaque domains is affected by mobility of the Cx proteins. These novel findings indicate that the GJ Nexus is a dynamic membrane organelle, with cytoplasmic and membrane-embedded proteins binding and diffusing according to distinct parameters.
Highlights
Gap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells
Abbreviations AQP4 Aquaporin 4 orthogonal particle arrays (OAPs) Orthogonal array of particles formed by AQP4 GJ Gap junction Cx Connexin Cx30 Connexin 30 Cx43 Connexin 43 EAAT2b Excitatory amino acid transporter 2b CC2-DMPE Coumarin phospholipid plmtGFP Palmitoylated GFP b5Ext B5Extended zonula occludens-1 (ZO-1) Zona occludens 1 Ocln Occludin EBFP2 Enhanced blue fluorescent protein 2 fluorescence recovery after photobleaching (FRAP) Fluorescence recovery after photobleaching GFP Green fluorescent protein HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid msfGFP Monomerized superfolder green fluorescent protein Regions Of Interest (ROI) Region of interest RT Room temperature sfGFP Non-monomerized superfolder green fluorescent protein GFP-Cx43tXXX Rat Cx43 tagged with GFP on the amino-terminus truncated at the indicated amino acid (i.e. GFP-Cx43t258 is truncated by mutagenesis of lysine 258 to a stop codon)
The localization of membrane-associated molecules with respect to Cx43 GJ plaques was examined by two-channel confocal microscopy and degree of overlap was quantified with Pearson correlation coefficients obtained from line scans along the junctional membrane (Fig. 1D, E)
Summary
Gap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells. Fluorescence recovery after photobleaching of tagged Nexusassociated proteins showed that mobility in plaque domains is affected by mobility of the Cx proteins These novel findings indicate that the GJ Nexus is a dynamic membrane organelle, with cytoplasmic and membrane-embedded proteins binding and diffusing according to distinct parameters. To gain insight into the dynamic relationship between Cx43 and other cellular components, we have determined the diffusivity and location of other molecules relative to the Cx43 plaque For these studies we selected for comparison small fluorescent lipids and lipid-tethered fluorescent proteins, the water channel Aquaporin[4] (AQP4) whose presence with Cx43 at astrocyte endfeet regulates ion homeostasis, a glutamate transporter (EAAT2b) that provides astrocytes with a mechanism to take up glutamate at active synapses, and junction-associated proteins (the other astrocyte gap junction protein Cx30, the tight junction proteins occludin and ZO-1)
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