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Selectivity of Connexin43 and Connexin40 Comprised Gap Junctions
Gap junctions are aggregates of intercellular channels each formed of protein subunits termed connexins (Cx). Recently published data show that junctional dye permeability relative to conductance (permselectivity) varies across several orders of magnitude for Cx43 junctions, suggesting variable selectivity of the comprising Cx43 channels. Logical candidates for this variable selectivity are variability in charge or size selectivity. Consequently, junctional charge and size selectivities were determined in the current study by simultaneous measurement of junctional permeance to dyes of differing size or charge.The results show that for a number of dyes differing in size, charge, chemical composition, and structure the primary determinant for selectivity through Cx43 gap junctions was the size of the dye permeant with this selectivity showing essentially no variability beyond that seen between incompletely divided cells, presumably representing the variability inherent to the measurement. As such, selectivity of dye-permeable Cx43 channels is well described by the physical dimensions of the channel pore acting essentially as a simple molecular sieve. The seemingly disparate dye selectivity and permselectivity results can be reconciled by the variable presence of a dye-impermeable but electrically conductive channel conformation for Cx43 channels, affording a possible mechanism for independent regulation of diffusion of larger molecules versus electrical conductance to smaller ions.Cx40 junctions, known to be cation selective, also showed minimal variability in charge selectivity indicating that Cx40 charge selectivity is also an essentially fixed parameter. Co-expression of Cx40 and Cx43 lead to charge selectivities ranging from Cx43 to Cx40 with an average intermediate between the two. Activation of PKC leads to an increase in cationic selectivity of Cx40/Cx43 composed junctions by specifically reducing permeability through non-selective Cx43 channels favoring permeation through cation-selective Cx40 channels, allowing for junctional charge selectivity regulation.The combined data suggest that selectivity properties for dye permeable channels composed of Cx43 or Cx40 are essentially fixed parameters of the channel pore. Only upon co-expression of these connexins is significant variability in selectivity seen. The differential effects of PKC-mediated phosphorylation on permeability of Cx43 and Cx40 channels then allows for regulation of junctional charge selectivity but only in cells expressing both connexins
Cardioprotective Role For the CLC-3 Chloride Channel in the mdx Mouse Model of Duchenne Muscular Dystrophy
Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43
Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (Bdye) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (−1). The ratio of dye rate constants (BNBD-M-TMA/BAlexa 350) indicated that Cx40 junctions are cation selective (10.7 ± 0.5), whereas Cx43 junction are nonselective (1.22 ± 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions
Selectivity of Connexin 43 Channels Is Regulated Through Protein Kinase C–Dependent Phosphorylation
Hindered Diffusion through an Aqueous Pore Describes Invariant Dye Selectivity of Cx43 Junctions☆
The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size or charge was determined in four cell types: Rin43, NRKe, HeLa43, and cardiac myocytes. The results show that Cx43 junctions are size- but not charge-selective and that both selectivities are constant parameters of junctional function. The consistency of dye selectivities indicates that the large continuum of measured junctional permselectivities cannot be ascribed to an equivalent continuum of individual channel selectivities. Further, the relative dye permeance sequence of NBD-M-TMA ∼ Alexa 350 > Lucifer yellow > Alexa 488 ≫ Alexa 594 (Stokes radii of 4.3 Å, 4.4 Å, 4.9 Å, 5.8 Å, and 7.4 Å, respectively) and the conductance sequence of KCl > TEACl ∼ Kglutamate are well described by hindered diffusion through an aqueous pore with radius ∼10 Å and length 160 Å. The permselectivity and dye selectivity data suggest the variable presence in Cx43-comprised junctions of conductive channels that are either dye-impermeable or dye-permeable