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Tejada MA
,
Stople K
,
Hammami Bomholtz S
,
Meinild AK
,
Poulsen AN
,
Klaerke DA
.
???displayArticle.abstract??? Slick (Slo2.1) and Slack (Slo2.2) channels belong to the family of high-conductance K+ channels and have been found widely distributed in the CNS. Both channels are activated by Na+ and Cl- and, in addition, Slick channels are regulated by ATP. Therefore, the roles of these channels in regulation of cell excitability as well as ion transport processes, like regulation of cell volume, have been hypothesized. It is the aim of this work to evaluate the sensitivity of Slick and Slack channels to small, fast changes in cell volume and to explore mechanisms, which may explain this type of regulation. For this purpose Slick and Slack channels were co-expressed with aquaporin 1 in Xenopus laevis oocytes and cell volume changes of around 5% were induced by exposure to hypotonic or hypertonic media. Whole-cell currents were measured by two electrode voltage clamp. Our results show that Slick channels are dramatically stimulated (196% of control) by cell swelling and inhibited (57% of control) by a decrease in cell volume. In contrast, Slack channels are totally insensitive to similar cell volume changes. The mechanism underlining the strong volume sensitivity of Slick channels needs to be further explored, however we were able to show that it does not depend on an intact actin cytoskeleton, ATP release or vesicle fusion. In conclusion, Slick channels, in contrast to the similar Slack channels, are the only high-conductance K+ channels strongly sensitive to small changes in cell volume.
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25347289
???displayArticle.pmcLink???PMC4210196 ???displayArticle.link???PLoS One
Figure 2. Sensitivity of Slack channels to changes in cell volume.Slack channels were co-expressed with AQP1 in Xenopus laevis oocytes and activated with a voltage protocol, as described in figure 1, under isotonic (A), hypotonic (B) and hypertonic media (C). (D) shows the corresponding I/V curves under isotonic (black), hypotonic (blue) and hypertonic (red) conditions. Control experiments for native, un-injected oocytes (UI, green) and oocytes only expressing AQP1 (violet) are also shown. In (E), maximal currents were measured at the end of the +80 mV depolarizing step under isotonic, hypotonic and hypertonic buffers and the currents were normalized to the current measured at isotonic conditions. Data points in panels (D) and (E) show the mean of 10 independent experiments ± S.E.M. (F) shows a current trace over time for a representative oocyte expressing Slack and AQP1. The channels were activated by a protocol as described in figure 1.
Figure 3. Kinetics of Slick channels during regulation by cell volume changes.(A) Slick channels (Slick, black) or Slick channels and AQP1 (Slick+AQP1, red) were expressed in Xenopus laevis oocytes and were kept at a holding potential of −80 mV before the channels were activated by depolarization to +80 mV (500 ms). The recorded currents were normalized to the maximal current measured in each experiment at the end of the +80 mV depolarization period for two representative oocytes. (B) oocytes expressing Slick channels in absence of co-expressed AQP1 were activated by depolarizations to +80 mV as before in isotonic (black), hypotonic (blue) and hypertonic (red) media, and currents were normalized to the maximal current. The figure shows the result of one representative experiment. (C) the experiments shown in (B) were repeated with oocytes co-expressing Slick channels and AQP1 channels (in isotonic (black), hypotonic (blue) and hypertonic (red) media). The figure shows the result of one representative experiment (Note: the currents measured in isotonic and hypotonic media are precisely superimposed). (D) a representative oocyte co-expressing Slick and AQP1 was exposed to isotonic (black), hypotonic (blue) and hypertonic (red) media and in each medium Slick channels were activated by a depolarization protocol as in figure 1. The figure shows the resulting IV-curves, each normalized to the maximal current (found at +80 mV).
Figure 4. Mechanisms for cell volume regulation of Slick K+ channels.Slick channels were co-expressed with AQP1 in Xenopus laevis oocytes and the channels were activated by depolarizations to +80 mV for 500 ms (holding potential: −80 mV). In all cases, the maximal currents (at the end of the depolarization period) were measured in isotonic, hypotonic and hypertonic media and are shown as relative changes in the current during cell swelling (hatched bars) and shrinkage (horizontal bars) as compared to the current under isotonic conditions (open bar). The figures show the effect of (A) cytochalasin D (10 µM, preincubation 24 hours), (B) Brefeldin A (10 µM, preincubation 8 hours), (C) Apyrase (5 U/ml) and (D) ATP (100 µM). All values are given as means ±SEM (n = 6–8).
Figure 1. Regulation of Slick channels by small changes in cell volume.In the upper panels, Slick channels were co-expressed with AQP1 in Xenopus laevis oocytes and were activated by a step protocol (−100 mV to +80 mV in 20 mV increments of 500 ms) from a holding potential of −80 mV (4 s). Currents were recorded in isotonic (A), hypotonic (B) or hypertonic (C) media. (D) shows the corresponding I/V relationships under isotonic (black), hypotonic (blue) and hypertonic (red) conditions. Control experiments for native, un-injected oocytes (UI, green) and oocytes only expressing AQP1 (violet) are also shown. In (E) maximal currents were measured at the end of the depolarization to +80 mV under isotonic, hypotonic and hypertonic buffers and the currents were normalized to the current measured at isotonic conditions. Data points in panels (D) and (E) show the mean of 10 independent experiments ± S.E.M. (F) displays a current trace over time for a representative oocyte expressing Slick and AQP1. The expressed Slick channels were activated by depolarization to +80 mV (500 ms) from a holding potential of −80 mV (3 s). The Figure shows the current measured at the end of the depolarization period as a function of time. Changes from isotonic medium to hypo- or hypertonic media are marked with arrows in the figure (the apparent delay from change in medium (arrows) to changes in the recorded currents reflect the “dead volume” in the flow system).
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