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    • When PKC was applied alone it attenuated the

    2021-11-10

    When PKC19–31 was applied alone, it attenuated the activity run-down following excision. Furthermore, the activity induced by addition of PKC19–31 during PMA treatment in many cases was higher than in controls. Accordingly, in our experimental conditions, the mechanism which underlays channel inhibition seems to work at a background level, in the absence of PKC activators, and this mechanism is augmented by PKC activation. Here, the picture emerging for IKCa channels of human erythrocytes is similar to the regulation of most of the structurally unrelated BKCa channels in smooth muscle, which are up-modulated by PKA and down-modulated by PKC [31]. Modulation of IKCa channels by PKC has been studied: (a) by measurements of Ca2+ pump transport activity and of K+ efflux in sickle red blood Ki16425 australia [16]; (b) by whole-cell current recording in CHO and HEK293 cells expressing the canine isoform cIK1 [14]; (c) by single channel recording in T84 cells expressing endogenous hIK1 channels [18] and (d) by measurements of 86Rb influx in normal and sickle human erythrocytes [15]. Fathallah et al. [16] found that incubation with PMA inhibited both Ca2+ pump and charybdotoxin-sensitive Ca2+-stimulated K+ efflux in sickle red blood cells. As far as cIK1 modulation is concerned, it has been reported that cIK1 currents are regulated in two ways by PMA, with an indirect acute activation and with a long-term inhibitory effect on the PKC site at T329, disclosed after overnight incubation with the drug. The authors suggest that this second effect might be due to altered recycling of cIK1 channels in the plasma membrane [14]. Devor and Frizzell [18] found that the inhibition of hIK1 channels of T84 cells by the PKC activator DIC8, previously reported [17], was not reproduced by treatment with the PKC catalytic subunit, concluding that PKC does not acutely regulate hIK1 channels in T84 cells. Rivera et al. [15] found that charybdotoxin-sensitive Rb influx in human erythrocytes was inhibited by PKC inhibitors and suggested that activation of Gardos channels by endothelin-1 might be mediated by PKC. The difference in the acute effects (inhibition vs. activation) of PKC on native hIKs and on cIK1s is remarkable. It should not be due to the technical approaches used by Wulf and Schwab [14] and by us, since both whole-cell and inside–out configurations of the patch-clamp technique involve cytoplasm dialysis. Also, the nature of these channels, native hIK in erythrocytes and heterologous cIK1 in CHO and HEK293 cells, does not seem to explain the differences of the acute effects of PKC activation. In fact, native cIK1 channels in canine MDCK cells are similarly activated by PKC [32]. Moreover, unlike hIK, cIK1 channels lack PKA sensitivity. Thus, it is likely that human and canine isoforms of IKCa channels are inserted in specific molecular assemblies, resulting in a PKC action that is opposite or synergistic with Ca2+, respectively, in controlling channel gating. This picture is in keeping with tissue-specific actions of other protein kinases on BKCa channels [31]. It is not possible with the available data to explain the results reported by Rivera et al. [15] for native human Gardos channels. They found a reduction of charybdotoxin-sensitive Rb influx in erythrocytes permeabilized to divalent ions with A23187 and preincubated with inhibitors of PKC. Since Rivera et al. did not activate PKC pharmacologically, in principle, their results might be induced by the several cellular mechanisms which are controlled by the increase of [Ca2+]i. In Rivera et al.'s [15] experiments PKCα should be translocated by high calcium but its activity might depend on indirect calcium-promoted phospholipase C activity. Our findings deal with a more delimited system and involve persistent PKC activation and its inhibition by exogenous drugs. On the other hand, our experimental conditions allow: (i) direct identification of IKCa channels; (ii) reversibility of the effects; (iii) rigorous control of the medium composition on both sides of the membrane; (iv) parsimonious use of pharmacological tools; (v) the preservation of integrated modulation by endogenous kinases and phosphatase in excised patches. In these conditions, the acute and reversible contribution of PKC to the channel modulation is a reduction of the opening frequency. In addition, we showed that increasing [Ca2+]i over 1 μM progressively masks the inhibitory effect of PKC modulation. Our findings are in agreement with the reduced Ca2+-stimulated K+ efflux in sickle cells reported by Fathallah et al. [16]. These authors used high [Ca2+]i and PMA to translocate and activate PKC and used 4αPDD as negative control, as in our experiments. Moreover, our data are in keeping with the observation that PMA treatment prevented deoxygenation-induced SS cell dehydration [33], making it unlikely that an excitatory net effect might be found when measurements are made on whole cells.