Calcineurin-dependent regulation of gap junction conductance and connexin phosphorylation in guinea pig left atrium

Figures

• 

  Fig. 1. Gap junction electrical properties in control and low-Na solutions. A Plots of calculated resistance (R) and reactance (-X) in the complex plane from an individual experiment in control solution (closed circles); low-Na solution (closed squares); washout to control solution (open circles). Fits to the data are made with a circle function with a centre of radius shifted along the R-axis (see Methods). The intercepts with the abscissa, R1 and R2, are used to estimate sarcoplasmic resistivity, Rc, and intracellular resistivity, Rj, from which gap junction resistivity, Rj, is derived. B Values of Gj (= 1/Rj) in control solution, in low-Na solution and after washout to control solution. The connecting bars are data from individual experiments. Horizontal bars in each data set show median values; note the logarithmic scale of the ordinate

• 

  Fig. 2. Calcineurin inhibitors (CysA and CAIP) on gap junction conductance, Gj, and Cx43 phosphorylation. A Gj values in control or low-Na solution, each in the absence or presence of cyclosporin-A (CysA, n = 6). Gj is expressed as a percentage of values in control solution. B As with part A but with the more specific calcineurin inhibitor, CAIP (n = 6). C: Representative Western blots for total Cx43 (T-Cx43, upper blot) and pSer368-Cx43 expression (lower plot) and also, in each case, GAPDH – see Methods for details. Lane 1 is molecular weight markers; Lanes 2 and 3 are from control tissue derived protein, lanes 4–9 lysates from low-Na treated tissues (lanes 6–7 and 8–9, are also plus CysA and CAIP, respectively). D: signal quantitation for pSer368-Cx43, normalised to T-Cx43. Data are mean ± SD, all n = 6. ***p < 0.001; low-Na (± calcineurin inhibitor) vs control: §§p < 0.01, low-Na vs low-Na + calcineurin inhibitor

• 

  Fig. 3. Tautomycin (TTM) and fostriecin (FST) on gap junction conductance, Gj, and Cx43 phosphorylation in normal and low-Na solution. A Effect of TTM. Left; Gj in low-Na solution as percentage of values in control and in the absence or presence of TTM (n = 6, left panel). Right; protein expression of pSer368-Cx43 normalised to total Cx43 (T-Cx43) in control and low-Na solution in the absence or presence of TTM (n = 6, right panel). B Effect of FST on the same experimental paradigm. ***p < 0.001; low-Na vs control: §§p < 0.01; §§§p < 0.001 low-Na vs low-Na + calcineurin inhibitor

• 

  Fig. 4. CaMKII and PKC inhibitors on gap junction conductance, Gj. A Effect on Gj of the CaMKII inhibitors KN93 (left) and AIP (right). In control and low-Na solution (both n = 6). B Effect on Gj of the PKC inhibitor chelerythrine (n = 5) ***p < 0.001; low-Na vs control

• 

  Fig. 5. Phosphorylation of serine and threonine Cx40 residues; effects of calcineurin and phosphatase inhibitors. A Representative Western blots for the detection of T-Cx40, pSer-Cx40 and pThr-Cx40 phosphorylation. Upper panel set: Lane 1, molecular weight markers; lanes 2–4, control tissue, lanes 5–10, low-Na treated tissues (lanes 8–10 also treated with CAIP). Lower panel set: Lane 1, molecular weight markers; lanes 2,3 control tissue, lanes 5–9, low-Na treated tissues (lanes 7,8 and 9,10 also treated with TTM and FST respectively). B Signal quantitation of pSer-Cx40 data normalised to T-Cx40 for control and low-Na data (in the additional presence of CysA, CAIP, FST and TTM). C: Signal quantitation of pThr-Cx40 data normalised to T-Cx40 as in part B. ***p < 0.001; low-Na vs control: §§p < 0.01; §§§p < 0.001 low-Na vs low-Na + CysA or FST

• 

  Fig. 6. Regulation of myocardial gap junction electrical conductance (Gj) by calcineurin. Gap junction (GJ) conductance is controlled by varying the phosphorylation of particular residues on connexin proteins (Cx43, Cx40) – two residues are shown (1, 2) one of which is phosphorylated (top GJ in diagram) to yield a high conductance channel. A rise of intracellular [Ca2+], [Ca2+]i, activates the phosphatase, calcineurin (Cn) to dephosphorylate residue-1 (step 1). This enables protein kinase-C (PKC) to phosphorylate residue-2, yielding a low conductance channel (step 2). Left atrial (LA) and left ventricular (LV) myocardium differ in the way Cn dephosphorylates residue-1. With LA tissue, data from this study are consistent with a direct action of Cn on residue-1, and partial intermediation of the phosphatase PP2A. With LV tissue, data (ref (12)) are consistent with dephosphorylation and deactivation of an inhibitor (I1) of the phosphatase PP1. Particular residues -1 and -2 on Cx43 are serine365 and serine368; but those on Cx40 involve unknown serine and threonine residues. The phosphatase inhibitors used are shown in italics under the relevant enzyme; see text for more details