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Am J Physiol Renal Physiol 293: F316-F324, 2007. First published April 25, 2007; doi:10.1152/ajprenal.00455.2006
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Lack of a role of membrane-protein interactions in flow-dependent activation of ENaC

Marcelo D. Carattino,1 Wen Liu,2 Warren G. Hill,1 Lisa M. Satlin,2 and Thomas R. Kleyman1,3

1Renal-Electrolyte Division, Department of Medicine, and 3Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania; 2Division of Pediatric Nephrology, Department of Pediatrics, Mount Sinai School of Medicine, New York, NewYork

Submitted 14 November 2006 ; accepted in final form 16 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rates of Na+ absorption in the distal nephron increase proportionally with the rates of tubular flow. We tested the hypothesis that the deformation or tension generated in the plasma membrane in response to flow activates the epithelial sodium channel (ENaC). We modified the physical properties of the membrane by changing the temperature and the content of cholesterol. Rates of net Na+ absorption measured in cortical collecting ducts (CCDs) perfused at room temperature at slow (~1) and fast (~5 nl·min–1·mm–1) flow rates were less than those measured at 37°C at the same flow rates, although increases in tubular fluid flow rates led to comparable relative increases in net Na+ absorption at both temperatures. Xenopus laevis oocytes expressing ENaC responded to an increase in shear stress at 22–25°C with a discrete delay followed by a monoexponential increase in whole-cell Na+ currents. We observed that temperature affected 1) basal currents, 2) delay times, 3) kinetics of activation, and 4) fold-increase in macroscopic currents in response to flow. The magnitude of the response to flow displayed biphasic behavior as a function of temperature, with a minimal value at 25°C. Steady-state fluorescence anisotropic measurements of purified plasma membranes did not show any obvious phase transition behavior over a temperature range from 8.3°C to 36.5°C. Modification of the content of membrane cholesterol did not affect the response to flow. Our results suggest that the flow-dependent activation of ENaC is not influenced by modifications in the intrinsic properties of the plasma membrane.

shear stress; mechanosensitive; mechanoregulation; anisotropy

THE EPITHELIAL SODIUM CHANNEL (ENaC) mediates the rate-limiting step of Na+ absorption across apical membranes of high-resistance Na+-transporting epithelia including the distal nephron, airway and alveoli, and distal colon. ENaCs are composed of three structurally related subunits termed {alpha}, beta, and {gamma}, which have intracellular NH2 and COOH termini and two membrane-spanning domains connected by large extracellular loops (30). In the distal segments of the nephron, rates of K+ secretion and Na+ absorption are regulated by the rates of intraluminal flow (21, 25, 26, 31, 33, 38, 43, 50, 57). Mechanisms that lead to an increase in ENaC activity in response to mechanical forces, such as laminar shear stress (LSS) or pressure (3, 10, 17, 18, 29, 37, 50, 53), have not been defined. We and others previously reconstituted flow-dependent activation of ENaC in the Xenopus laevis oocyte expression system and showed that the response to flow reflects a rapid increase in the open probability of the channel (3, 10). Two alternative models have been proposed to explain mechanisms of activation of mechanosensitive channels in response to mechanical stimuli: 1) the bilayer model and 2) the tethered model (28). The bilayer model proposes that tension or deformation developed in the bilayer in response to a mechanical force directly gates the channel. The tethered model proposes that mechanical forces are transmitted directly to the channel through intracellular and/or extracellular tethers (28). We observed that Cys substitutions in the area adjacent to and including the selectivity filter of the {alpha}-subunit of ENaC (tract Ser580-Ser589) determine significant changes in the time course of channel activation by LSS, suggesting that this area experiences conformational changes during flow-dependent activation (11). Based on the close proximity of these residues in the pore region of ENaC with the plasma membrane, it is possible that ENaC responds to LSS by sensing the deformation or tension generated in the plasma membrane during flow stimulation.

Biological membranes are capable of deforming in response to external stresses (e.g., erythrocytes in blood capillary) or through association with the cytoskeleton (41). Recent studies have demonstrated the importance of lipid-protein interactions in the gating of prokaryotic mechanosensitive channels (48). Prokaryotic mechanosensitive channels have been proposed to sense deformations in the bilayer profile, including the extent of hydrophobic mismatch defined as the difference between the hydrophobic length of the transmembrane protein structures and the hydrophobic thickness of the membranes that they span, and the intrinsic curvature of the membrane leaflet (47). In mammalian cells, activation of the epithelial Na+/H+ exchanger by hyposmotic stress is caused by the curvature imposed on the membrane on cell swelling (2). Temperature and cholesterol content are recognized to influence the curvature, thickness, bending elasticity, and fluidity of membranes (13, 41, 45, 54). The fluidity of the membrane, which is related to the extent of disorder and molecular motion within the plasma membrane, regulates the activity of membrane-bound proteins, enzymes, ion channels, receptor-associated protein kinases, and sensor proteins (36). In endothelial cells, temporal and spatial changes in membrane fluidity are observed in response to an increase in fluid shear stress (9, 27). These changes in membrane fluidity are likely to affect the regulation of membrane proteins (9). For example, stimulation of endothelial cells by fluid shear stress, hypotonic stress, or a membrane-fluidizing agent leads to conformational changes and activation of the bradykinin B2 G protein-coupled receptor (12).

In this report, we evaluated the hypothesis that ENaC is activated directly by the deformation or the tension generated in the plasma membrane following increases in rates of flow, by means of physical and chemical approaches that disturb the intrinsic properties of the membrane. Our results suggest that ENaC is not activated directly by the changes developed in the plasma membrane in response to flow.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Female New Zealand White rabbits obtained from Covance (Denver, PA) were housed in the Mount Sinai School of Medicine Center for Comparative Medicine. Rabbits were allowed free access to food and water. Adult female X. laevis were purchased from Xenopus Express (Plant City, FL). Animals were euthanized in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animal protocols were approved by Institutional Animal Care and Use Committees at the Mount Sinai School of Medicine and the University of Pittsburgh.

Oocyte expression. cRNAs for {alpha}-, beta-, and {gamma}-mENaC subunits were synthesized with T3 mMessage mMachine (Ambion, Austin, TX). Stage V-VI X. laevis oocytes were pretreated with 1.5 mg/ml type II or IV collagenase and injected with 0.3–1 ng of cRNA/subunit. Injected oocytes were maintained at 18°C in modified Barth's saline [MBS; (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 15 HEPES, 0.3 Ca(NO3)2, 0.41 CaCl2, and 0.82 MgSO4, pH 7.4] supplemented with 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, and 100 µg/ml gentamicin sulfate.

Two-electrode voltage clamp. Two-electrode voltage clamp (TEV) was performed using a GeneClamp 500B amplifier (Axon Instruments, Union City, CA) as previously described (10). Temperature was determined with a probe localized in close proximity to the oocyte with a TC-10 temperature controller (Dagan, Minneapolis, MN). The temperature of the solutions was regulated by a system of recirculating water lines. Data were acquired through Clampex 8.0 and stored on the hard drive of a computer. Pipettes filled with 3 M KCl had resistances of 0.5–5 M{Omega}. The extracellular solution (TEV solution) was (in mM) 110 NaCl, 2 KCl, 1.54 CaCl2, and 10 HEPES, pH 7.4. The recording chamber was perfused at a rate of 3.5 ml/min during the experiment. LSS was applied by perfusing TEV solution through a vertical pipette localized above the oocyte surface at a rate of 1.6 ml/min (10). A single experiment was performed with each oocyte, and the bath temperature was kept constant during the experiments with a precision of ±1°C. ENaC-mediated whole-cell Na+ currents were defined as the benzamil-sensitive component of the current. To determine the time course of activation by LSS, oocytes were clamped at a holding potential of –60 mV and stimulated by perfusion through a vertical pipette with a computer-controlled protocol generated in pClamp 8.0. Following the stimulation process, the ENaC-mediated component of the whole-cell Na+ current was determined by bath perfusion with TEV solution supplemented with 5 µM benzamil.

Microperfusion of single tubules for measurement of net cation transport rates. Kidneys were removed via a midline incision, and single tubules were dissected freehand in cold (4°C) Ringer solution containing (in mM) 135 NaCl, 2.5 K2HPO4, 2.0 CaCl2, 1.2 MgSO4, 4.0 lactate, 6.0 L-alanine, 5.0 HEPES, and 5.5 D-glucose, pH 7.4, 290 ± 2 mosmol/kgH2O, as previously described (57). A single tubule was studied from each animal. Isolated CCDs were microperfused in vitro as previously described (44), and tubular fluid samples were collected at both a slow physiological (~1 nl·min–1·mm–1) and fast (~5 nl·min–1·mm–1) flow rates. The sequence of flow rates was randomized within each group of tubules to minimize any bias induced by time-dependent changes in ion transport. Tubules were perfused and bathed at either room temperature or 37°C with Burg's perfusate containing (in mM) 120 NaCl, 25 NaHCO3, 2.5 K2HPO4, 2.0 CaCl2, 1.2 MgSO4, 4.0 Na+ lactate, 1.0 Na3 citrate, 6.0 L-alanine, and 5.5 D-glucose, pH 7.4, 290 ± 2 mosmol/kgH2O (57, 58). During the 60-min equilibration period and thereafter, the perfusion chamber was continuously suffused with a gas mixture of 95% O2-5% CO2 to maintain pH of the Burg's solution at 7.4 at room temperature and at 37°C. The bathing solution was continuously exchanged at a rate of 10 ml/h using a peristaltic syringe pump (Razel, Stamford, CT). Collected samples were analyzed for Na+ concentrations by helium glow photometry, and the rates of net Na+ absorption were calculated, as previously described (44, 57, 58).

Fluorescence anisotropy on isolated oocyte plasma membranes. Plasma membranes from 400 X. laevis oocytes were prepared using a modification of the method outlined elsewhere (37). Briefly, oocytes were homogenized using a Maxima homogenizer (Fisher Scientific, Pittsburgh, PA) in R-buffer (10 µl/oocyte) containing (in mM) 250 sucrose, 1 CaCl2, 1 MgCl2, 20 Tris·HCl, pH 7.4, and Complete Mini Protease Inhibitors (Roche Diagnostics, Mannheim, Germany). The homogenate was centrifuged twice at 500 g for 5 min, and the supernatant was recovered each time. The supernatant was diluted 1:1 with an equal volume of 50% Optiprep in R-buffer, placed in a centrifuge tube, and 16 ml of a 20%-0% Optiprep gradient in R-buffer was poured on top of the membranes. The gradients were centrifuged in a swinging bucket rotor at 50,000 g for 90 min. Assays of alkaline phosphodiesterase activity (a plasma membrane marker) on fractions taken from the top third of the Optiprep gradient reveal an ~90-fold enrichment of plasma membrane compared with homogenate (data not shown). Membranes taken from the gradient were diluted three-fold then concentrated by pelleting at 100,000 g for 30 min and resuspended in 300 µl buffer. Membranes were incubated with 2 µM 1,6-diphenyl-1,3,5-hexatriene (DPH) for 30 min at room temperature in the dark. Fluorescence polarization measurements were conducted on an Aminco Bowman Series 2 luminescence spectrometer equipped with a magnetically stirred and water-jacketed cuvette holder (for temperature regulation) and automated prism polarizers. Polarized fluorescence measurements were made starting with the cuvette solution equilibrated at ~6°C, every 10 s at excitation and emission wavelengths of 354 and 428 nm, respectively. After 8–10 measurements, data acquisition was paused and the temperature of the circulating water bath was increased 2- 3°C, and after a 10-min equilibration period data collection resumed. The ratio of polarized fluorescence was then used to calculate anisotropy by AB2 software (Spectronic Unicam). Briefly, the sample was excited by vertically polarized light, and the vertical and horizontal emitted fluorescence, Ivv and Ivh, were used to calculate anisotropy. Values of steady-state fluorescence anisotropy reflect the degree of motion exhibited by the fatty acyl chains within the bilayer. These measurements report the degree to which the linear probe has rotated during the lifetime of its excited state. In a more fluid membrane, the degree of rotation is higher (anisotropy is lower) because excursions of acyl chains from some initial spatial configuration are greater. Thus these values can be taken as an index of fluidity and membrane order.

Cholesterol depletion. Cholesterol depletion was performed as previously described by Sadler and Jacobs (49). Each oocyte was incubated in 1 ml of MBS either with or without 5 mM methyl-beta-cyclodextrin (MbetaCD; Sigma-Aldrich, St. Louis, MO) for 1 h at 18°C. To estimate cholesterol depletion, groups of 60 oocytes were incubated in 2 ml of MBS containing 1 mg/ml BSA (MBS-BSA) and 5 µCi of [3H]-cholesterol (specific activity 57.6 Ci/mmol; PerkinElmer, Boston, MA) for 30 min, and washed two times with 10 ml of MBS-BSA and two times with 30 ml of MBS. Oocytes were separated in groups of 20 and incubated in 10 ml of MBS with or without 5 mM MbetaCD for 1 h at 18°C. After the incubation, oocytes were washed six times with 10 ml of MBS, moved to a glass vial, and solubilized in 500 µl of TS-2 tissue solubilizer (Research Products International, Mount Prospect, IL) at 60°C. Before liquid scintillation spectrometry was performed, 150 µl of 10% acetic acid and 10 ml of Scientisafe 30% (Fisher Scientific, Hampton, NH) were added to each vial.

Cholesterol enrichment. The content of cholesterol was increased by incubation of the oocytes with water-soluble cholesterol (C-4951, Sigma-Aldrich). Cholesterol solutions (6.9 mg/ml) prepared in MBS were filtered through a 0.22-µm filter. The final concentration of cholesterol in the solution was estimated with a colorimetric cholesterol assay (Cholesterol E kit, Wako, Osaka, Japan). Each oocyte was incubated in 1 ml of MBS either with or without 0.4 mM cholesterol for 1 h at 18°C. To estimate cholesterol enrichment, groups of 20 cells were incubated in 10 ml of MBS containing 0.1 µCi/ml of 3H-cholesterol and 0.4 mM water-soluble cholesterol for 1 h at 18°C. Oocytes were washed six times with 10 ml of MBS, transferred to a glass vial, and solubilized by addition of 500 µl of TS-2 tissue solubilizer (Research Products International). Before liquid scintillation spectrometry, 150 µl of 10% acetic acid and 10 ml of Scientisafe 30% (Fisher Scientific) were added to the scintillation cocktail mixture.

Total content of cholesterol. Groups of 70 oocytes were homogenized with 150 µl of a solution containing 110 mM NaCl, 2 mM KCl, and 10 mM HEPES, pH 7.4. Homogenates were centrifuged at 500 g for 5 min, and supernatants were recovered and centrifuged at 20,000 g for 3 h at 4°C. Pellets containing the membranes were resuspended in 1 ml of a mixture of hexane:isopropanol (3:2), rocked for 1 h to extract lipids, and centrifuged at 20,000 g for 15 min at 4°C. Supernatants were transferred to a glass tube and dried down under a stream of nitrogen at 60°C. Total cholesterol was quantified with a colorimetric method (Wako).

Fluorescence anisotropy on intact oocytes treated with cholesterol-modifying reagents. Groups of 30 uninjected oocytes were treated with either 1) 5 mM MbetaCD to deplete plasma membrane cholesterol, 2) 0.4 mM water-soluble cholesterol to augment plasma membrane cholesterol, or 3) were placed in MBS at 18°C for 1 h. At the end of 1 h, they were washed three times with ice-cold MBS and then were incubated at 4°C for 30 min in the presence of 10 µM DPH in MBS. Oocytes were washed three times in cold MBS, then homogenized in a volume of 1 ml MBS in a sintered glass mortar with sintered glass pestle (15 strokes). Oocyte homogenates were clarified by centrifugation at 5,000 g for 5 min, and then 200 µl of crude membranes was measured for fluorescence anisotropy at room temperature.

Data and statistical analyses. Data are expressed as means ± SE (n), where n equals the number of independent experiments analyzed. Data were analyzed with Clampfit 9.0 (Axon Instruments) or SigmaPlot 8.02 (SPSS, Chicago, IL). The time constant of activation ({tau}) was determined by fitting experimental data with an exponential function (1)

Formula
where I is the macroscopic current, {tau} is the time constant, and a and c are constants determined by curve fitting. For experiments performed at 17, 30 and 37°C, the time course of activation was fit with the following equation

Formula
where I is the macroscopic current, {tau}1 and {tau}2 are time constants, and a1, a2, and c are constants determined by curve fitting. The delay time ({delta}) was estimated as the time between the delivery of the stimulus and the intersection of the exponential phase with the baseline current. For experiments performed at 25°C, {delta} was estimated using the following equation (2)

Formula
where I0 is the current at time 0 before stimulation. For experiments performed at 17, 30 and 37°C, {delta} was determined as the time at which the initial change in current occurred by inspection of the recordings. Statistical comparisons were performed using GraphPad Instant (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Temperature influences several properties of biological membranes, including curvature, thickness, bending elasticity, and degree of motion exhibited by the fatty acyl chains within the bilayer (fluidity). If the regulation of ENaC by flow involves sensing the deformation or tension generated in the plasma membrane, changes in the physical properties of the plasma membrane should influence the response. We examined the temperature dependence of the flow response of ENaC in both rabbit cortical collecting tubules and ENaC-expressing X. laevis oocytes. Oocytes are usually studied at room temperature, whereas Na+ transport in isolated, perfused rabbit tubules is measured at 37°C. We previously reported that the fold-increase in Na+ transport in response to flow is significantly greater in collecting tubules (~4- to 5-fold at 37°C) than in oocytes expressing ENaC (~0.5-fold at 22–25°C), when subjected to similar levels of LSS (9, 10, 43, 50). We confirmed that an increase in tubular fluid perfusion rate from 1.0 ± 0.1 to 5.5 ± 0.2 nl·min–1·mm–1 led to an increase in net Na+ absorption from 14.9 ± 1.8 to 71.4 ± 19.0 pmol·min–1·mm–1 (n = 7; P < 0.03; Fig. 1) in CCDs perfused at 37°C. In CCDs perfused at room temperature, the rate of net Na+ absorption at the slow flow rate of 1.1 ± 0.1 nl·min–1·mm–1 (4.1 ± 2.6 pmol·min–1·mm–1; n = 5) was significantly less than that measured in segments perfused at 37°C (P < 0.01). An increase in tubular fluid flow rate in the CCDs studied at room temperature to 5.5 ± 0.3 nl·min–1·mm–1 was associated with an increase net Na+ absorption (to 26.7 ± 12.1 pmoll·min–1·mm–1) that approached statistical significance (P < 0.08). The fold-increase in the net rate of Na+ absorption in response to an increase in flow from low (~1 nl·min–1·mm–1) to high (~5 nl·min–1·mm–1) rates was similar at both room temperature (4.3 ± 2.3-fold) and 37°C (5.0 ± 1.1-fold; P = 0.78, unpaired t-test). In perfused CCDs, previous work has shown that modifications in the temperature of incubation induced significant changes in the fluidity of the membrane measured by trimethylammonium-diphenylhexatriene anisotropy (23), indicating that properties of the membrane should be modified under our experimental conditions. Our results of in vitro perfused CCDs suggest that flow-dependent activation of ENaC is not affected by the modifications induced by temperature.


Figure 1
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  		Fig. 1. Effect of temperature on flow stimulation of net Na+ absorption in perfused cortical collecting ducts (CCDs). Rates of net Na+ absorption (top) and transepithelial voltage (bottom) measured in CCD tubules perfused at low (1 nl·min–1·mm–1) or high (5 nl·min–1·mm–1) flow rates at room temperature or at physiological temperatures (37°C). Each data point represents the means ± SE of 5–7 tubules. Student's t-test with the Welch correction was used for comparisons.

 
We previously demonstrated that ENaC expressed in X. laevis oocytes respond in a dose-dependent manner to changes in rates of LSS, with a rapid increase in the open probability (10). We evaluated the effects of temperature on the time course of activation of ENaC by LSS. Oocytes expressing wild-type ENaC were subjected to stimulation by LSS at temperatures varying between 17 and 37°C. The ratio of the peak current following activation by flow to basal current (I/I0) showed a strong dependence with regard to temperature (Figs. 2 and 3). I/I0 plotted as a function of temperature displayed biphasic behavior with a minimal value at 25°C (1.48 ± 0.07, n = 27) and a maximal at 37°C (3.11 ± 0.30, n = 19). At 17°C (1.84 ± 0.10, n = 15) and at 30°C (1.74 ± 0.05, n = 22) the response was intermediate (Fig. 3). Correlation analyses between basal and peak currents revealed that peak currents following activation by LSS were strongly correlated with basal currents for experiments performed at 17 (r2 = 0.944), 25 (r2 = 0.925), 30 (r2 = 0.968), and 37°C (r2 = 0.918). This suggests that either the basal current determines the magnitude of the response or another variable(s) influences both basal and LSS-stimulated currents (Fig. 4).


Figure 2
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  		Fig. 2. Representative tracings of activation of epithelial Na+ channels (ENaC) by laminar shear stress (LSS) at various temperature. Wild-type mouse {alpha}beta{gamma}-ENaC-expressing oocytes were perfused with two-electrode voltage clamp (TEV) solution, and a computer-controlled perfusion system was used to activate fluid-jet perfusion via a vertical pipette (at time 0). Currents were monitored at a holding potential of –60 mV. The gray lines represent fitting of the recordings by a monoexponential (25°C) or biexponential (17, 30, 37°C) equation as described in MATERIALS AND METHODS.

 

Figure 3
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  		Fig. 3. Effect of the temperature on the activation of Na+ currents by LSS. Wild-type ENaC-expressing oocytes were perfused with TEV solution, and benzamil-sensitive Na+ currents were determined before and during fluid-jet perfusion. The peak response of the whole-cell benzamil-sensitive Na+ current following the initiation of LSS was normalized to the basal current (I/I0) at different temperatures. The magnitude of the response at 25°C was significantly lower compared with the response at 17 (P < 0.05), 30 (P < 0.05), and 37°C (P < 0.001) by Kruskal-Wallis test, nonparametric ANOVA, following by Dunn's multiple comparisons test. Experiments were performed with 15–27 oocytes/group.

 

Figure 4
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  		Fig. 4. Correlation analyses of flow-dependent activation of ENaC at different temperatures. The peak responses of the whole-cell benzamil-sensitive Na+ currents following the initiation of LSS were plotted as a function of the basal benzamil-sensitive Na+ currents. The coefficient of determination obtained by Pearson's correlation analysis is indicated by r. Experiments were performed with 15–27 oocytes/group.

 
The time course of flow-dependent activation of ENaC was dramatically influenced by temperature. A change in the temperature from 25°C led to significant changes in the parameters of activation including 1) a decrease in the delay time ({delta}) between the delivery of the LSS stimulus and the increase in whole cell Na+ current; 2) a modification of the time course of channel activation including the order of the exponential function describing the activation (monoexponential vs. biexponential activation); and 3) a modification in the absolute magnitude of the current increase (I/I0) (Figs. 2 and 3 and Table 1). The time course of activation at 17, 30, and 37°C was best described by a biexponential function supporting our previous observation that activation of ENaC by LSS involves multiple steps (Table 1).


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  		Table 1. Effects of temperature on parameters of ENaC activation by LSS

 
Our results in oocytes demonstrate that the response of ENaC to LSS displays a biphasic profile as the temperature is changed from 17 to 37°C. If the effects of the temperature on the flow-dependent activation of ENaC in oocytes are mediated by a direct modification of the properties of the plasma membrane, we might expect the fluidity of the membrane to display a biphasic change with temperature. While it is not possible to measure fluidity directly with steady-state fluorescent anisotropy, the technique does provide a measure of lipid order since the probe (DPH) is more sensitive to order (half-cone angle of motion) than to the rate of probe rotation (membrane viscosity) (32, 34). We studied the changes in the steady-state fluorescence anisotropy as a function of temperature in purified plasma membranes from X. laevis oocytes. The membrane order profile at different temperatures is shown in Fig. 5. The relationship between the two parameters was relatively linear. There was no evidence of any abrupt phase transitions, suggesting that the biphasic behavior in flow-dependent activation of ENaC is not due to temperature-specific perturbations in membrane order. These results suggest that the changes in the flow-dependent activation of ENaC observed in oocytes at various temperatures are not associated with the modifications in the fluidity of the membrane.


Figure 5
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  		Fig. 5. Order/fluidity behavior of oocyte plasma membranes as a function of temperature. Fluorescence anisotropy of isolated oocyte plasma membranes equilibrated with 2 µM 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured at different temperatures as described in MATERIALS AND METHODS. Data shown are representative of 4 experiments utilizing plasma membranes of oocytes from 4 individual frogs.

 
We used a second approach to alter the fluidity and other properties of the membrane by removing or exogenously adding cholesterol. Cholesterol content influences several properties of model membranes and lipid extract bilayers, including fluidity, thickness, bending elasticity, and curvature (13, 41, 45, 54). In bovine endothelial cells, cholesterol depletion or enrichment affects the activation of volume-regulated anion channels by an osmotic stimulus, suggesting that cholesterol content modulates the deformation of the membrane (35). To remove cholesterol from the membrane, we used an approach previously described by Sadler and Jacobs (49) which takes advantage of the cholesterol-sequestering properties of MbetaCD. We confirmed that our protocols for depleting (or supplementing) membrane cholesterol were effective. The reduction in the content of cholesterol measured with this protocol was 25.4 ± 2.2% (n = 8) (see MATERIALS AND METHODS). To increase the content of cholesterol in the membrane, oocytes expressing ENaC were incubated for 1 h in MBS supplemented with 0.4 mM (water-soluble) cholesterol. The amount of cholesterol incorporated per oocyte using [3H]-cholesterol as a marker was 0.100 ± 0.006 µg (n = 9) (see MATERIALS AND METHODS). The total content of cholesterol estimated in the membrane fraction was 0.295 ± 0.026 µg/oocyte (n = 6). To evaluate the effect of cholesterol on membrane order profile, we measured fluorescence anisotropy on intact membranes from oocytes treated with cholesterol-modifying reagents. The membrane order profile for control, cholesterol-depleted, and cholesterol-enriched membranes is shown in Fig. 6. Membrane order significantly decreased in cholesterol-depleted membranes and increased in cholesterol-enriched membranes compared with control membranes.


Figure 6
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  		Fig. 6. Cholesterol content does not affect the response of ENaC to flow. Wild-type ENaC-expressing oocytes were incubated for 1 h in modified Barth's saline (MBS) containing either vehicle control (MBS), 5 mM methyl-beta-cyclodextrin (MbetaCD), or 0.4 mM cholesterol. A: effect of membrane cholesterol depletion and enrichment on flow-activated Na+ currents. Benzamil-sensitive Na+ currents were determined before and during fluid-jet perfusion via a vertical pipette. The peak response of the whole-cell benzamil-sensitive Na+ current following the initiation of LSS was normalized to the basal current (I/I0). Experiments were performed with 13–16 oocytes/group. B: order/fluidity behavior on cholesterol depletion and enrichment of oocyte plasma membranes. Fluorescence anisotropy on intact oocytes treated with cholesterol-modifying reagents was measured as indicated in MATERIALS AND METHODS. Values are means ± SE for 5–6 anisotropy determinations. The results are representative of 3 separate experiments. Statistical significance was determined by Student's t-test (*P < 0.05, **P < 0.01).

 
The magnitude of the response of ENaC to LSS was similar in either control (1.58 ± 0.15, n = 16), cholesterol-depleted (1.50 ± 0.07, n = 13) or cholesterol-enriched oocytes (1.54 ± 0.10, n = 13) (P = 0.74, n = 13–16, Kruskal-Wallis test, nonparametric ANOVA) (Fig. 6). Delay times (P = 0.57, ANOVA) and time constants of the exponential activation (P = 0.93, ANOVA) were similar in control, cholesterol-depleted, and cholesterol-enriched oocytes (Table 2).


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  		Table 2. Effects of cholesterol content on parameters of ENaC activation by LSS at 25°C

 
Experiments with control, cholesterol-depleted, and cholesterol-enriched oocytes were performed with the same batches of oocytes. In these set of experiments, the delay times (P < 0.0001, unpaired t-test) and time constants (P < 0.0001, unpaired t-test with Welch correction) of the exponential activation were significantly reduced compared with experiments performed at 25°C (11) and reported in Table 1. Within individual batches of oocytes, the measured parameters of ENaC activation by LSS appear to be consistent. The differences in these parameters noted in Tables 1 and 2 likely reflect batch-to-batch oocyte variability.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Geometric transformations of the membrane are required to gate prokaryotic mechanosensitive channels (48). We examined whether the plasma membrane plays a role in the flow-dependent activation of ENaC. Temperature variation was used as an approach to influence the properties of the membrane such as the curvature, fluidity, thickness, bending elasticity, and physical state. We found that temperature reduction led to a decrease in absolute rates of Na+ absorption and in transepithelial voltage in rabbit CCDs (Fig. 1). However, the fold-increase in rates of Na+ absorption when luminal flow was increased from 1 to 5 nl·min–1·mm–1 was similar at room temperature and 37°C, suggesting that the flow-dependent activation of ENaC is not influenced by the properties of the plasma membrane. It was previously demonstrated in CCDs that an increase in the temperature of the perfusion solution was associated with an increase in the fluidity of the plasma membrane (decrease in trimethylammonium-diphenylhexatriene anisotropy) (23), supporting the fact that temperature affects the membrane of in vitro perfused CCDs. The decrease in the rates of basal Na+ absorption observed in experiments performed at room temperature could be explained, in part, by a reduction in the single-channel conductance of ENaC. In cortical collecting tubules, ENaC single-channel conductance is reduced from 9 to 5 pS as temperature is decreased from 37 to 22°C (46). In addition, a fraction of the reduction of rates of net Na+ absorption detected in CCDs perfused at room temperature could be due to a decrease in basolateral Na+-K+ pump activity. We consider this possibility unlikely as 1) immunocytochemical studies show that basolateral distribution of Na+-K+-ATPase remains unchanged during cold exposure (to 4 h) followed by rewarming (8); and 2) the rates of ouabain-inhibitable basolateral 86Rb uptake, a functional index of Na+-K+ pump activity, in rabbit CCDs maintained at room temperature for 1 h were identical to those measured in tubules maintained at 37°C (16). Studies performed in oocytes (discussed below) suggest that temperature affects ENaC open probability (4, 5, 14, 15), although these results were not corroborated in cortical collecting ducts.

The effects of the temperature on the steady-state current of ENaC expressed in oocytes have been extensively studied (4, 5, 14, 15). At a macroscopic level in X. laevis oocytes expressing ENaC, channel activity displays a graded response to temperature with maximal activity at 6°C and half-maximal response at ~25°C (4). Studies of the effect of temperature on single-channel properties of ENaC in oocytes demonstrated that single-channel conductance decreased by about half, whereas channel open probability increased between two- to threefold when the temperature of the perfusion was decreased from 30 to 15°C (14). Changes in temperature have opposing effects on rates of Na+ transport in CCDs and X. laevis oocytes. A decrease in temperature from 37 to 25°C was associated with a marked reduction in rates of basal Na+ absorption in CCDs, but an increase in basal Na+ current in oocytes. These results indicate that Na+ transport under basal conditions in CCDs and X. laevis oocytes expressing ENaC is regulated either by different modulator/s or by the same modulator/s that exhibits different sensitivity to temperature in these two systems. The four- to fivefold increase in net Na+ absorption in response to an increase in flow rates observed in perfused CCDs suggests that the open probability of ENaC at low perfusion rates must be lower than 0.2, with the consideration that the stimulation by flow could lead to an increase in the open probability to a value close to 1, which is probably an overestimation. The magnitude of the response to flow seems to correlate with the activity of the channel under basal conditions in both oocytes and CCDs. Differences in the activity of the channel under basal conditions in oocytes and CCDs may be due to several factors, including posttranslational modifications such as phosphorylation/dephosphorylation and proteolysis and differences in interaction with regulatory proteins and modulators.

In oocytes expressing ENaC, temperature influences the time course of the response to LSS by changing delay times, time constants of activation, order of the exponential function describing the activation (monoexponential vs. biexponential), and maximal response. Temperature has been shown to influence other processes that affect ENaC activity. For example, ENaC activity is influenced by the extracellular Na+ concentration ([Na+]), a process referred to as Na+ self-inhibition (6, 7, 15, 24, 51). This phenomenon is observed following a fast change from an extracellular solution containing a low [Na+] to a high [Na+]. ENaC-mediated Na+ currents are rapidly increased as the external [Na+] is rapidly elevated until it reaches a peak, which is followed by an exponential decrease to a steady-state level. The ratio between the peak and steady-state current reflects the magnitude of the Na+ self-inhibition response. The ratio between the peak and the steady-state Na+ current as well the steady-state current itself are temperature dependent (15). Both Na+ self-inhibition and flow-dependent regulation of ENaC reflect changes in channel open probability. The effects of temperature on the kinetics of both processes likely reflect the temperature dependence of channel transitions during processes of activation and/or inhibition.

As a general paradigm, the gating of ion channels involves conformational changes of the protein that are influenced by temperature. In oocytes expressing ENaC, changes in perfusate temperature affect both delay times and activation time constants in response to flow. We previously observed that the time course of activation by LSS involves several steps (11). It is not surprising that some of these steps display significant temperature dependence, which could reflect a modification of the properties of one or more components of the channel complex. The effects of the temperature on the kinetics of ENaC activation by LSS may reflect changes in several steps, including 1) sensing of the mechanical stimulus, 2) transmission of the stimulus from the mechanosensor to the channel's gate, and 3) changes in conformation of the gate that allow for ions to cross the channel.

The degenerin/ENaC superfamily of ion channels is composed of members expressed in tissues as diverse as lung, kidney, and nervous systems of nematodes, flies, and vertebrates (30). In the nematode Caenorhabditis elegans, degenerins are expressed in touch-sensitive neurons, where they play a role in the detection of gentle body touch (39). Degenerins are thought to constitute the pore-forming subunits of the mechanotransducing complex, being tethered to its intracellular and extracellular domains (39). Although the channel-forming subunits are tethered intracellulary to MEC-7 and MEC12, which encode beta- and {alpha}-tubulins, respectively, is unlikely that microtubules play a role in mechanosensation (20). Mutations in at least three extracellular matrix proteins, MEC-5, which encodes a unique collagen, and MEC-1 and MEC-9, which encode multidomain proteins with several epidermal growth factor domains and several Kunitz-like domains, are associated with a defective touch phenotype, suggesting that channels are gated directly by forces transmitted through the extracellular matrix (19, 20).

Properties of the lipid bilayer, including thickness, intrinsic curvature of the membrane, and bending elasticity are thought to be influenced by temperature and cholesterol content (13, 41, 45, 54). Our results indicate that the mechanosensitivity of ENaC is not dependent on sensing of the deformation or tension generated in the plasma membrane by flow and suggest, alternatively, that the large extracellular domains of ENaC subunits might play a key role in sensing mechanical forces. In an analogous manner to the mechanosensory complex in C. elegans, a complex of proteins might be associated with the extracellular domains of the channel and have a role in the sensing and transduction of the mechanical stimulus to the pore-forming subunits of the channel. The extracellular matrix is a complex structural entity surrounding and supporting cells in multicellular organisms. In vertebrates, the extracellular matrix is composed of structural proteins (collagen, elastin), associated and specialized proteins (e.g., fibrillin fibronectin, laminin, thrombospondin, tenascin), and glycosaminoglycans and proteoglycans (1). Current reports suggest that endothelial cell glycocalyx acts as a mechanotransducer that senses shear stress in blood vessels (22, 42, 55, 56). In CCDs, the organization of the extracellular matrix is largely unknown. Given the structural complexity of the extracellular matrix, it is likely that its components and organization differ in oocytes and CCDs, accounting for part of the differences observed in the response to flow in these systems.

In summary, our data suggest that the response of ENaC to flow is not directly influenced by the properties of the plasma membrane. Interactions have been demonstrated between ENaC and components of the cytoskeleton (40, 52), but not between ENaC extracellular domains and components of the extracellular glycocalyx. Components of the ENaC complex, other than the three pore-forming subunits, that are required for ENaC to elicit a response to flow remain to be identified.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38470 and DK-51391.


   ACKNOWLEDGMENTS
 
Present address of W. G. Hill: Dept. of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, 840 Memorial Dr., Cambridge, MA 02139.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. R. Kleyman, Renal-Electrolyte Div., Univ. of Pittsburgh, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (e-mail: kleyman{at}pitt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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