"Are dye and electrical coupling properties of Cx43 comprised gap junctions independently regulated?"

ABSTRACT
Recently published data indicate for both positively and negatively charged dyes that permselectivity (dye permeability relative to conductance Pdye/gj) of Cx43 junctions varies over two orders of magnitude (Circ.Res. 98:1498, 2006; Biophys J 91:565, 2006). For the cationic dye, Pdye/gj variability was reduced from a 151 to 6-fold range when phosphorylation at serine 368 in the carboxy-terminus (CT) of Cx43 was prevented. These observations indicate that the permselectivity of Cx43 channels varies consequent to phosphorylation-dependent regulation by the cell. To ascertain whether variable permselectivity is explained by variable charge or size selectivity of the comprising channels, one cell of a pair of Cx43 expressing Rin cells (Rin43) was injected simultaneously with two dyes, either Alexa350 and NBD-M-TMA (negative vs. positively charged, similar size) or Alexa 350 and Alexa 594 (both negatively charged, limiting diameters of ~5 vs. 12 angstroms), and the relative rates (kdye) of transjunctional diffusion for both dyes determined. Results from junctions and cytoplasmic bridges are compared to discern junction-specific differences. The kNBD/kAlexa350 ratio for Cx43 junctions and cytoplasmic bridges was 1±0.1. The kAlexa350/kAlexa594 ratio for junctions and bridges were 35±3 and 3.5±0.4, respectively. These data indicate that Cx43 junctions are size but not charge selective; the absence of variability in these ratios indicates that variable permselectivity is not explained by variable charge or size selectivity. Since regulation of permselectivity involves phosphorylation of the CT, we examined Cx43-M257 (truncated at residue 257) to determine whether removal of this domain altered selectivity. Data from the truncated channel did not differ from the wildtype for either dye combination. These data suggest that Cx43 channels exist in at least two configurations: one that is poorly permeated by dyes (the major determinant of junctional conductance) and one that is well-permeated by dyes (the major determinant of dye permeability).