Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels.
The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (gammaj) of about 20, 40-45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the gammaj distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [32P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D018076:DNA, Complementary / Q000096:biosynthesis; D019922:GAP-43 Protein; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D014162:Transfection
D000596:Amino Acids; D018076:DNA, Complementary; D019922:GAP-43 Protein; D007473:Ion Channels; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C016276:8-bromocyclic GMP; D011494:Protein Kinases; D006152:Cyclic GMP