Electrical conductance of mouse connexin45 gap junction channels is modulated by phosphorylation

In this study we report about the modulation of connexin45 (Cx45) gap junct ion channel properties by phosphorylation of the connexin molecules through different protein kinases. Phosphorylation of Cx45 was studied in HeLa cel ls transfected with mouse Cx45 (mCx45). Using Western blotting (WB) and imm unocytochemistry, these cells were found exclusively positive for Cx45 and the protein was separated as a doublet of bands with a calculated mass of 4 6 and 48 kD. After dephosphorylation using calf intestine phosphatase (CIP) , the 48 kD band disappeared almost completely leaving a single band at 46 kD. This effect can be prevented by including phosphatase inhibitors during CIP treatment. These results indicate that the 48 kD signal represents a p hosphorylated form of Cx45. To investigate the effects of (de)phosphorylati on of Cx45 on the conductive properties of gap junction channels built of t his connexin, cell pairs were subjected to dual voltage clamp experiments a nd coupling was determined before and after addition of PMA, 4 alpha-PDD, c AMP, cGMP and pervanadate to the superfusate. 100 nM of the PKC activating phorbol ester PMA increased normalized junctional conductance by 50.9+/-28% . 100 nM of the inactive phorbol ester 4 alpha-PDD had no significant effec t. Activation of PKA with 1 nM 8-Br-cBMP decreased coupling by 20.9+/-5.7% while 1 mM 8-Br-cCMP (PKG-activation) was ineffective. 100 mu M pervanadate , a tyrosine phosphatase inhibitor, reduced coupling by 43.7+/-11.1%. Singl e channel measurements, under identical phosphorylating conditions, were no t significantly different from each other and all frequency histograms exhi bited two conductance peaks at approximately 20 and 40 pS. WB analysis reve aled, as compared to control conditions, a relative increase of the 48 kD s ignal upon stimulation with pervanadate (142+/-42%) and 8-Br-cAMP (50+/-23% ) whereas neither stimulation with PMA nor 8-Br-cGMP had a significant effe ct. These experiments show that electrical intercellular conductance via Cx 45 gap junction channels is differentially regulated by phosphorylation. Ho wever, regulation does not act by changing single channel conductance, but most likely by modulation of the open probability of Cx45 gap junction chan nels. (C) 2000 Elsevier Science B.V. All rights reserved.