Browsing by Author "FISHMAN, GI"

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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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    HUMAN CONNEXIN43 GAP JUNCTION CHANNELS - REGULATION OF UNITARY CONDUCTANCES BY PHOSPHORYLATION
    (1994) MORENO, AP; SAEZ, JC; FISHMAN, GI; SPRAY, DC
    Connexin43 is the major gap protein in the heart and cardiovascular system. Single channel recordings of human connexin43 gap junction channels exogenously expressed in transfected SKHep1 cells demonstrate two discrete classes of channel events, with unitary conductances of predominantly 60 to 70 and 90 to 100 pS when recorded with an internal solution containing CsCl as the major current-carrying ionic species and at moderate transjunctional voltages (<60 mV). Human connexin43 expressed in SKHep1 cells displays multiple electrophoretic mobilities (apparent M(r), approximate to 41 to 45 kD) when resolved in Western blots. Treatment of connexin43 from these cells with alkaline phosphatase collapses the bands into a single 41-kD species; application of alkaline phosphatase to the cell interior through patch pipettes yields channels that are predominantly of the larger unitary conductance. The smaller 60- to 70-pS unitary conductance values correspond to the most common channel size seen in cultured rat cardiac myocytes; these channels were more frequently observed after treatment with the phosphatase inhibitor okadaic acid, which was shown to increase phosphorylation of human connexin43 in these cells under similar conditions. Exposure to the protein kinase inhibitor staurosporine shifted the proportion of events toward the largest unitary conductance and resulted in decreased phosphorylation of human connexin43 in seryl residues in these cells. Thus, the unitary conductance of human connexin43 gap junction channels covaries with the phosphorylation state of the protein. This change in unitary conductance appears to be a unique effect of phosphorylation on gap junction channels, since it has not been observed for other ion channels that have thus far been evaluated.