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Figure 1. . Cooling reversibly stimulates hENaC in Xenopus oocytes. Shown is an example of the reversible protocol (see text). (A) Decreasing temperature from 23°C stimulated conductance and capacitance (data corrected for the amiloride-insensitive conductance). (B) Expanded scale of the changes of Cm, gNa, and temperature demonstrating a lag between bath temperature changes and the effects on hENaC. (C) Scatter plot of Cm versus gNa demonstrating that the majority of conductance changes occurred before the bulk changes of Cm. (D) Scatter plot of bath temperature versus gNa demonstrating that the stimulation of gNa lags the temperature changes. (E) Scatter plot demonstrating an initial decrease of gNa if bath temperature was decreased below 10°C. Additional examples using a sequential temperature change (see the protocol in Fig. 3) indicated the absence of a secondary stimulation of gNa at temperatures below ∼15°C.
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Figure 2. . Summary of the temperature-induced changes in hENaC expressing oocytes. The amiloride-sensitive conductance exhibited a biphasic relationship in response to decreasing temperature. These effects were fit with two linear relationships with slopes of −0.099 and 0.091, leading to a Q10 of −1.92 and 1.83 at high and low temperatures, respectively. These relationships intersected at a temperature of 15.2°C, defined as the maximal temperature or Tmax. A small stimulation of Cm is observed. This stimulation was not different than that observed in control oocytes (see Fig. 3), indicating no specific effects on ENaC-expressing oocytes. Data normalized to the values at 25°C that averaged 11.7 ± 1.6 mS and 0.233 ± 0.013 mF for g Na (amiloride-sensitive component) and Cm, respectively. n = 9.
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Figure 3. . Effects of temperature on control oocytes. (A and B) Representative effects on the capacitance and conductance. Cooling caused a small stimulation of Cm and a rapid decrease of gm. The decrease of conductance was monotonic, in contrast to that observed in hENaC-expressing oocytes. (C and D) Summary of the changes induced by temperature. (C) The decrease of conductance could be fit with a single exponential function with a constant of 6.2°C. (D) The increase of Cm was ∼10% at 15°C, and was similar to that observed with ENaC (Fig. 2). This small increase is unlikely related to trafficking, but rather to the effects of temperature on the membrane dielectric coefficient (see text). Data are normalized to the values at 25°C or control which averaged 2.6 ± 0.5 mS and 0.218 ± 0.012 mF for gm and Cm, respectively. n = 6.
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Figure 4. . Summary of the temperature-induced changes in hENaC expressing oocytes preequilibrated in the low Na+ solution (9.4 mM Na+, Na+ substituted with NMDG). This results in an intracellular Na+ activity in the range of 7 mEq (Awayda, 1999). Under these conditions, similar effects of temperature were observed as in Fig. 2, indicating the lack of effects of intracellular and extracellular [Na+] on the biphasic stimulation of gNa, and the small increase of Cm. The biphasic relationships observed were fit with lines with slopes of −0.081 and 0.069, leading to a Q10 of −1.86 and 1.50 at high and low temperatures, respectively. This resulted in a slightly shifted Tmax of 12.8°C; similar to that observed in high Na+. Data normalized to the values at 25°C which averaged 42.1 ± 11.4 mS and 0.217 ± 0.014 mF for gNa and Cm, respectively. n = 8.
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Figure 5. . Representative effect of temperature on the whole cell currents and voltage activation in a hENaC-expressing oocyte. Oocytes were held at 0 mV and clamped in increments of 20 mV from −100 to +40 mV for a period of ∼1,600 ms. Note the activation of currents at negative voltages at 15°C, followed by inhibition at 4°C. Amiloride was added at the beginning and end of the experiment. Voltage activation was fit as described by Awayda (2000) to the current response to a voltage command of −100 mV (most negative current shown). Note the change in time constant with decreasing temperature, and the increase in the magnitude of activation (ΔIV) at 15°C followed by marked inhibition at 4°C.
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Figure 6. . Summary of effects of temperature on voltage activation. Experiments were performed in oocytes bathed in ND94. (A) A large increase of t is observed with decreasing temperature leading to ∼5-fold increase at 10°C. (B) A biphasic stimulation of the magnitude of voltage activation (ΔIV) is observed with a Tmax in the range of that calculated for gNa (see Fig. 2). (C) A similar biphasic stimulation is also observed with the initial current at −100 mV (I−100), and (D) with the ratio of ΔIV to I−100. Values at 4°C were eliminated as the time constants were >1,500 ms and could not be accurately fit from the acquired data points. n = 7.
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Figure 7. . Effects of temperature on oocyte membrane infolding. Oocytes were isolated, fixed, and processed for electron microscopy as described in materials and methods. Representative micrographs of oocytes incubated at 15°C (A), 20°C (B), and 25°C (C) for 30 min. PM denotes plasma membrane and its infolding as observed in cross section. A slightly higher degree of membrane folding is observed in (C). These findings are summarized in (D), and indicate a slightly lower infolding and presumably area in membranes from oocytes incubated at 15°C and 20°C as compared with those at 25°C. These changes are opposite to those expected from the effects on Cm, and indicate that the stimulation observed with ENaC is unrelated to increased membrane area but possibly attributed to dielectric coefficient changes (see text for additional details). n = 4.
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Figure 8. . Effects of temperature on oocyte membrane lipids. Oocyte membrane vesicles were isolated as described in materials and methods. (A) DPH-HPC fluorescence in response to decreasing temperature from 30°C to 5°C. Ihv and Ihh correspond to the fluorescent emission intensities in the vertical and horizontal directions in response to horizontal excitation. These are used to calculate an instrument and wavelength correction factor. Ivv and Ivh correspond to the vertical and horizontal emission intensities in response to vertical excitation. These values, along with the correction factor are used to calculate anisotropy. Note the biphasic nature of the effects of temperature on these intensities. (B and C) Calculated anisotropy also revealed biphasic effects of temperature. Data representative of five experiments.
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Figure 9. . Representative effects of gadolinium on an ENaC-expressing oocyte. (Top) Addition of 10, 50, and 100 μM Gd+3 caused a small and time dependent decrease of Cm. These effects were poorly reversible and were unlikely representative of actual area changes (see text). (Bottom) Effects on membrane conductance. As previously observed, the majority of the conductance is attributed to ENaC and is furthermore amiloride sensitive. Sequential addition of Gd+3 at 10, 50, and 100 μM caused a slow decrease of the amiloride sensitive conductance. The majority of these effects were observed with 10 μM Gd+3 and were time dependent in that very little additional changes were observed by increasing the [Gd+3]. Both effects were only partially reversible within the 1-h washout period. Data representative of nine experiments. See Table I for summary.
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Figure 10. . Representative example of the effects of chlorpromazine on ENaC-expressing oocytes. See Fig. 9 legend for additional details. Addition of CPZ at 10, 50, and 100 μM caused a slow decrease of the amiloride-sensitive conductance. CPZ also caused an accompanying decrease of capacitance. As observed with Gd+3, the changes of gm were larger than those of Cm. These effects were partially reversible upon washout of CPZ. Unlike Gd+3, the majority of the effects of CPZ were not observed until [CPZ] of 50 and 100 μM (see Table II for more details). Data representative of nine experiments.
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Figure 11. . Potential dielectric changes with Gd+3 and CPZ. Sequential addition of CPZ and Gd+3 caused only small additive changes of Cm, indicating a common mechanism of action. Subsequent addition of PMA further decreased Cm by ∼50%, similar to that previously reported (Awayda, 2000). However, this decrease was smaller than that which is expected from an intact membrane. These findings along with others (see text) indicate effects of CPZ and Gd+3 on the membrane dielectric properties rather than area. Note the rapid small decrease of conductance observed after Gd+3 addition in oocytes pretreated with CPZ. This may indicate facilitation of the action of Gd+3 in membranes where lipid order is already altered by the presence of CPZ. This was similar to the facilitation observed by altering temperature (see Figs. 13 and 14, and discussion). Data representative of five experiments.
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Figure 12. . Lack of effects of Gd+3 and CPZ on membrane area. Summary of membrane infolding in oocytes treated with 50 μM Gd+3 or 50 μM CPZ (see Fig. 7 for additional details). Note the absence of any decrease of membrane infolding after Gd+3 or CPZ treatment. This further supports the hypothesis that the changes of Cm observed with temperature, Gd+3, and CPZ are unrelated to changes of area, but rather reflect the changes of the membrane dielectric properties.
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Figure 13. . Cooling traps Gd+3 in the membrane. Representative effects of a temperature change on the actions of Gd+3. A decrease of bath temperature to 4°C altered the inhibitory time course of Gd+3. This temperature change also rendered Gd+3 completely irreversible by presumably locking its interaction with the plasma membrane. See text for additional details. Similar effects were also observed at 10°C and with CPZ (not depicted).
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Figure 14. . Changes of Gd+3 blocking kinetics at low temperatures. Gd+3 was added to oocytes precooled to 4°C. In these experiments, the inhibitory effects on gm were immediate (within the first 5 min), while the effects on Cm were eliminated. This provides further evidence for interactions between the membrane and Gd+3. n = 7.
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Figure 15. . Representative effects of Gd+3 and CPZ on membrane anisotropy. Vesicles were isolated as described in materials and methods. Arrows indicate experimental changes. (A) Addition of Gd+3 caused a slow time-dependent decrease of anisotropy. This decrease was not dose dependent in that similar effects were observed with 50 and 100 μM Gd+3. As these experiments used DPH-HPC, which is an outer leaflet specific probe, these changes reflect effects of Gd+3 on the outer membrane leaflet. Data representative of five experiments. (B) Addition of CPZ caused a decrease of membrane anisotropy with a much faster time course than that observed with Gd+3. Data representative of four experiments. (C) Preincubation of vesicles at 4°C prevented the actions of Gd+3 on membrane anisotropy. These effects are consistent with those observed in Fig. 14 demonstrating the elimination of the effects of Gd+3 on Cm and the altered blocking kinetics of gm, in oocytes precooled to 4°C. These findings are taken as further support of the membrane actions of Gd+3. Data representative of three experiments.
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