Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • br Materials and methods br Results

    2021-09-22


    Materials and methods
    Results Unitary currents of Gardos SB 415286 australia were identified by their conductance value, inward rectification, voltage independence of open probability, kinetics and Ca2+-sensitivity, all features which have been described elsewhere [8], [13], [20], [23], [24], [25], [26]. Because of the pipette shape and the sealing procedure used, membrane patches consisted of a large membrane area which was imaged using a fluorescent calcium indicator (Fig. 1A). Cell-free inside–out patches presumably preserved complex cytoskeletal structure and enzyme machinery, as indicated by the consistent presence of modulatory activity by kinases and phosphatases. Furthermore, unrestricted access to the internal face of the ion channels was allowed, as indicated by the quick interruption of activity when perfused by a low-calcium solution (Fig. 1B). The typical activity pattern of Gardos channels has been reported in a previous paper [13]. Briefly, in the cell-attached configuration, the channel activity was consistently low, because of the low calcium permeability and of the large-capacity Ca2+ pump of the human erythrocyte membrane. The activity rapidly increased after excision, when the free calcium concentration in the solution perfusing the inner side of the patch was higher than 0.2 μM. A spontaneous reduction of activity (run-down) occurred within minutes after excision. Channel activity was reversibly restored under perfusion of the membrane patch with a PKA-activating cocktail, containing 1 mM cAMP, 1 mM MgATP and 1 mM theophylline. Current up-modulation was mainly due to an increase of the opening frequency and was proportional to calcium concentration. These results demonstrated that the ion channels are up-regulated by an endogenous PKA, perhaps intimately associated with the channel itself. On the other hand, the occurrence of channel run-down both after excision and after interruption of perfusion with the stimulating cocktail was evidence for ongoing dephosphorylation reactions, which down-regulate channel activity [13]. Accordingly, in this study the effects of PKC activation were analyzed either before activity run-down or after PKA activation. The perfusion of freshly excised inside–out patches with the PKC activator PMA strongly reduced the open probability, in a reversible mode. Fig. 2 illustrates such an effect. At the beginning of the record, corresponding to the excision, openings increased in frequency because the calcium content of the perfusing solution was 1 μM. The time course of the mean patch current (Im) during two consecutive stimulations with PMA (2 μM) is displayed. The mean value of Im was reduced from 3.65 to 0.26 pA with a lag time of about 20 s. Wash-out was followed by an increase of activity, within about 15 s, while readmission of PMA induced a new inhibition. Repetitive stimulation never showed changes in patch responsiveness. In seven experiments, addition of PMA decreased Im to 17.3±7.7% of the control value, while it was restored to 106.5±14.1% after wash-out. The delay in getting a clear-cut inhibitory response to the PKC activator was widely variable from patch to patch. Two main factors were found to affect this delay. First, long lag times (minutes) were found to be associated with multichannel patches in which the distribution of the times spent at different conductance levels clearly deviated from the binomial distribution, suggesting that modulation differentially affected neighbouring ion channels. This aspect clearly needs further investigation, although it is beyond the scope of the present study. As far as the second factor is concerned, calcium concentration in the perfusing solution were found to affect both lag times and potency of channel inhibition. Channel activity was almost completely abolished by PMA in 0.5 μM Ca2+, it was reduced to less than 20% of the control activity in the Ca2+ range 1–2 μM, with delays increasing to minutes in 2 μM Ca2+, whereas the inhibition was undetectable in 10 μM Ca2+.