ENaC-membrane interactions: regulation of channel activity by membrane order

Abstract

Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459-6463). As these changes of temperature affect membrane lipid order and lipid-protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order-dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on gNa were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2 degrees C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd+3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd+3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC.

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 C_m, g _Na, and temperature demonstrating a lag between bath temperature changes and the effects on hENaC. (C) Scatter plot of C_m versus g _Na demonstrating that the majority of conductance changes occurred before the bulk changes of C_m. (D) Scatter plot of bath temperature versus g _Na demonstrating that the stimulation of g _Na lags the temperature changes. (E) Scatter plot demonstrating an initial decrease of g _Na 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 g _Na at temperatures below ∼15°C.

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 T_max. A small stimulation of C_m 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 C_m, respectively. n = 9.

Figure 3.

Effects of temperature on control oocytes. (A and B) Representative effects on the capacitance and conductance. Cooling caused a small stimulation of C_m and a rapid decrease of g_m. 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 C_m 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 g _m and C_m, respectively. n = 6.

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 g_Na, and the small increase of C_m. 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 T_max 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 g_Na and C_m, respectively. n = 8.

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 (ΔI_V) at 15°C followed by marked inhibition at 4°C.

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 (ΔI_V) is observed with a T_max in the range of that calculated for g _Na (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 ΔI_V 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.

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 C_m, 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.

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. I_hv and I_hh 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. I_vv and I_vh 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.

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 C_m. 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.

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 g _m were larger than those of C_m. 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.

Figure 11.

Potential dielectric changes with Gd⁺³ and CPZ. Sequential addition of CPZ and Gd⁺³ caused only small additive changes of C_m, indicating a common mechanism of action. Subsequent addition of PMA further decreased C_m 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.

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 C_m observed with temperature, Gd⁺³, and CPZ are unrelated to changes of area, but rather reflect the changes of the membrane dielectric properties.

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).

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 g _m were immediate (within the first 5 min), while the effects on C_m were eliminated. This provides further evidence for interactions between the membrane and Gd⁺³. n = 7.

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 C_m and the altered blocking kinetics of g _m, 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.