Browsing by Author "HERTZBERG, EL"

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    EFFECTS OF CGMP DEPENDENT PHOSPHORYLATION ON RAT AND HUMAN CONNEXIN43 GAP JUNCTION CHANNELS
    (1995) KWAK, BR; SAEZ, JC; WILDERS, R; CHANSON, M; FISHMAN, GI; HERTZBERG, EL; SPRAY, DC; JONGSMA, HJ
    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 (g(j)) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas g(j) was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g(j) 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 (gamma(j)) 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 gamma(j) 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[P-32] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of P-32] into human Cx43 was not affected. We conclude that 8Br-cCMP 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 g(j), which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.
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    ON THE MECHANISMS OF CELL UNCOUPLING INDUCED BY A TUMOR PROMOTER PHORBOL ESTER IN CLONE-9 CELLS, A RAT-LIVER EPITHELIAL-CELL LINE
    (1993) BERTHOUD, VM; ROOK, MB; TRAUB, O; HERTZBERG, EL; SAEZ, JC
    It is known that in Clone 9 (C9) cells, intercellular gap junctional communication (IGJC) is rapidly blocked by the tumor promoter phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), but it recovers spontaneously a few hours later and becomes refractory to TPA (Yada et al., J. Membr. Biol. 88, 217-232 (1985)). We now report that gap junctions between C9 cells contain at least two junctional proteins, connexin26 (Cx26) and connexin43 (Cx43), and that the TPA-induced changes in IGJC correlate temporally to changes in the state of phosphorylation of Cx43. The latter changes were prevented by inhibition of protein kinase C. Phosphoamino acid analysis and two-dimensional tryptic peptide maps of P-32-labeled Cx43 showed that during the TPA-induced phosphorylation at least two of the phosphorylated forms of Cx43 were differentially phosphorylated in seryl residues as compared to control. TPA induced a drastic reduction in junctional conductance as well as a redistribution of unitary gap junction channel event sizes seen in control cells. These changes were associated with retrieval of Cxs from the plasma membrane. Reappearance of gap junctions formed by Cx43 but not by Cx26 accounted for the spontaneous recovery in IGJC. It is proposed that gap junctions between C9 cells contain two types of channels each formed by Cx43 or Cx26 and that they are differentially affected during the action of TPA.