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Figure 2. ENaC currents are insensitive to osmotic cell swelling. Currents are summarized at a holding voltage of â100 mV. In this group of experiments, the baseline current was stable during the 30 min control period (compare the values of â30 min and 0 min in the 210 mOsm solution). The currents at 30 min after cell swelling (+30 min, 140 mOsm) were elevated above the iso-osmotic control values (0 min). However, this increase of current with cell swelling was likely due to the accompanying increase of the amiloride-insensitive current (compare 10 μM amiloride in 210 mOsm and 140 mOsm). n = 6.
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Figure 3. Effect of mechanical cell swelling on ENaC whole-cell currents. Representative example testing the effects of 90- and 180-nl increases of cell volume. The baseline ENaC currents are shown in A, and the amiloride insensitive currents are shown in B. A 90-nl increase of cell volume (see text) did not result in appreciable immediate (1 min, C) or long term (10 min, D) changes of current. A total increase of cell volume by 180 nl, by a second 90-nl injection 10 min after the first one, was still without immediate (1 min, Eâ) or long term (10 min, Fâ) effects on currents.
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Figure 4. ENaC currents are insensitive to mechanical cell swelling. Currents are summarized at a holding voltage of â100 mV. There was a small (<10%) increase of current 1 min after the first injection. This increase was transient and currents decayed to values expected of control. Similar results were observed in oocytes injected twice for a total cell-volume increase of 180 nl. This transient stimulation was similar to, but smaller than, that observed in control oocytes pretreated with cytochalasin B (Figs. 7 and 8). Therefore, it is likely unrelated to ENaC stimulation. Thus, ENaC currents were insensitive to both osmotic and mechanical cell swelling. n = 6 for the first 90-nl injection and n = 4 for the second 90-nl injection.
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Figure 7. Effect of mechanical swelling on whole-cell currents in ENaC and water-injected oocytes pretreated with cytochalasin B. Currents in ENaC-expressing oocytes (A) responded to injection of 90 nl of 100-mM KCl with a transient stimulation (10 s, B) that gradually declined toward baseline by 10 min (Câ). However, the same pattern of stimulation was observed in water-injected oocytes (D) that exhibited an initial increase (10 s, Eâ) that declined toward baseline within 10 min (Fâ). The two examples shown here were chosen to reflect the potential for very large initial stimulation in response to volume injection in both control oocytes and ENaC-expressing oocytes.
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Figure 8. Mechanical cell swelling activates an endogenous current in cytochalasin B-pretreated oocytes. ENaC-expressing oocytes responded by a transient increase of current at â100 mV 20 s after the 90-nl volume increase (A). However a similar response was observed in ENaC-expressing oocytes treated with 10 μM amiloride (B). This response was also observed in control oocytes (Câ) and is, therefore, unrelated to the presence of ENaC. The initial stimulation is summarized at 20 s rather than 10 s, as this value was not measured in all groups of oocytes. However, the stimulation at 20 s was only slightly lower than that observed at 10 s. n = 7 for each group.
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Figure 5. Effect of hypo-osmolarity on ENaC whole-cell currents in oocytes pretreated with cytochalasin B. Representative ENaC currents in oocytes pretreated with cytochalasin B (A) were not altered after 30 min of hypo-osmotic cell swelling (B). In this group of oocytes, a 25% decrease of osmolarity was the maximum decrease that could be tolerated before oozing.
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Figure 6. ENaC currents are also insensitive to osmotic cell swelling in the presence of cytochalasin B. Oocytes were pretreated with cytochalasin B for 2â5 h. Currents are summarized at a holding voltage of â100 mV. In this group of experiments there were no differences between the 0 min control currents recorded in the 210-mosmol solution and +30 min currents recorded 30 min after incubation in the 160-mosmol solution. There were also no differences between the amiloride-insensitive currents in oocytes treated with cytochalasin B and those untreated (see 10 μM amiloride in Fig. 4). n = 6.
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Figure 9. Effect of hyperosmolarity on ENaC currents. ENaC-expressing oocytes (A) responded to a 45-min incubation in 260 mosmol hyperosmotic solution by a decrease of whole-cell currents (B). The magnitude of the shrinking-inhibitable currents is shown in C and is indistinguishable from ENaC currents, and is, moreover, amiloride sensitive, as indicated by the relatively small values of amiloride-insensitive currents observed at the end of the experiment (D).
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Figure 10. Time-course of inhibition of amiloride-sensitive slope conductance after hyperosmotic cell shrinking. Data were corrected for the value of the amiloride-insensitive slope conductance as described in materials and methods and are plotted normalized to the conductance immediately before the hyperosmotic solution change. The time-control data were obtained from oocytes treated in a similar manner except that they were not incubated in the hyperosmotic solution. It is clear that a 25% increase of solution osmolarity caused a gradual decrease of ENaC conductance. This decrease of conductance was not observed in the time-control group. n = 6 and 7 for the hyperosmotic and time-control groups, respectively. *P < 0.001 and **P < 0.004 using a nonpaired Student's t test. All other data points were not significantly different from each other when compared between the two groups of oocytes at the same time interval.
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Figure 11. Effect of hyperosmolarity on ENaC currents in cytochalasin B-pretreated oocytes. ENaC expressing oocytes pretreated with cytochalasin B for 2â5 h (A) were not responsive to a 45-min incubation in 260 mosmol hyperosmotic solution (B). Indeed, the shrinking-inhibitable currents (Câ) were much smaller than those observed in the absence of cytochalasin pretreatment (Fig. 9 Câ) and may be explained by the small baseline drift observed during the control period (see Fig. 12). The amiloride-insensitive currents recorded at the end of the experiment were not affected by cytochalasin pretreatment (D).
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Figure 12. Time course of the changes of the amiloride-sensitive inward slope conductance in response to cell shrinking in cytochalasin B-pretreated oocytes. See Fig. 10, legend, for details. In this group of oocytes, cell-shrinking did not cause a large decrease of ENaC conductance as observed in the absence of cytochalasin B (Fig. 10). Moreover, there were no significant differences between the experimental and time-control groups of data. n = 6 and 7 for the hyperosmotic and time-control groups, respectively.
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Figure 13. Effect of a 5-in. H2O pipette pressure difference on ENaC single channel activity. Representative single channel records from a cell-attached patch in an ENaC-expressing oocyte demonstrating the lack of effect of applied pipette pressure. Patch voltage was â80 mV (inside the cell with respect to ground or bath). Upward deflections indicate channel openings. The open probability under control conditions (A) was 0.35. Application of a negative (B) or positive (Câ) pipette pressure of 5 in. H2O did not significantly alter the open probability. Conditions were as described in materials and methods.
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Figure 14. Time course of the changes of the single channel open probability in response to changes of pipette pressure. Open probability data were binned into 10-s intervals and were plotted as a continuous function of time. Despite the noticeable spontaneous changes of Po that were observed in the control period (0 in. ÎP) and toward the end of the experiment (+5 in. ÎP), there were no changes between the control period and each of the experimental periods, indicating lack of sensitivity of ENaC to either positive or negative pipette pressure difference.
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Figure 15. Summary data indicating the lack of effect of a 5 in. H2O pipette pressure difference on ENaC's open probability. As observed from the individual experiment data (A), there were no reproducible effects of negative or positive pipette pressure on Po. Examination of the Po in individual experiments as illustrated in Fig. 14 indicated that none of these experiments exhibited any measurable changes of activity immediately after applying either the positive or negative pipette pressures. The data from these nine experiments are summarized in B and also indicate the lack of significant effect of pipette pressure difference on the channel's mean open probability (P > 0.3 between all four groups).
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Figure 16. Effect of a 10-in. H2O pipette pressure difference on ENaC single channel activity. This larger pressure difference was also without effect on ENaC activity. However, an endogenous channel was stimulated by the application of +10 in. H2O despite the presence of blockers of endogenous channels in the pipette (see materials and methods). This channel could be easily differentiated from ENaC given the large difference in their kinetics. Conditions were the same as described in Fig. 13.
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Figure 17. Summary data indicating the lack of effect of a 10-in. H2O pipette pressure difference on ENaC's open probability. See text and Fig. 15, legend, for details.
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