Role of intracellular sodium concentration in osmotic-dependent regulation of mENaC expressed in Xenopus laevis oocytes

 

Galizia Luciano 

 Correspondence to:

Dr. Luciano Galizia

e-mail: lgalizia@gmail.com

 

Running head: Intracellular sodium affects ENaC osmotic response

 

Keywords:

ENaC

Oocytes

Hypotonic stimuli

 

 

Total number of Figures: 5

 

 

ABSTRACT   

 

            It has been previously reported that hypotonic stimuli can influence Na⁺ currents through the mouse epithelial sodium channel (ENaC) expressed in Xenopus laevis oocytes. Here we studied the influence of the intracellular sodium concentration ([Na⁺]_i) on the modulation of ENaC by hypotonic stimuli by measuring amiloride-sensitive Na⁺ currents (INa_amil). We were capable of controlling the [Na⁺]_i of mENaC-injected oocytes by exposing them to incremental concentrations of sodium in the extracellular medium. In addition, we found a bell-shaped relationship between [Na⁺]_i and ENaC activity expressed as inward conductance (GNa_inward), with a maximum near 20 mM. In these conditions, the rapid inhibitory effect of inward INa_amil to a mild hypotonic shock were consistently reduced at [Na⁺]_i values were GNa_inward was increased. Moreover, this relationship between increased conductance and decreased sensitivity to osmotic stimuli was independent of ENaC conductance levels. This result was obtained by injecting increasing masses of cRNA. Also, a direct effect of [Na⁺]_i during the experiment was discarded since acute sodium loads after 24-hours incubation in a low sodium concentration condition was not sufficient to reduce ENaC sensitivity to the osmotic shock. When we analyzed the kinetics of the ENaC currents during the hypotonic challenge at different [Na⁺]_i, we obtained a dependence by the extracellular sodium concentration [Na⁺]_o in the inactivation rate of currents during the hyperpolarized pulses. The presented data allows us to suggest the possibility that ENaC sensitivity to hyposmotic stimuli in Xenopus laevis oocytes is dependent on the intracellular sodium concentration in an indirect way.

 

1.       INTRODUCTION

 

            The epithelial Na⁺ channel (ENaC) is the rate limiting step for Na⁺ reabsorption across several epithelial tissues, including colon, sweat glands, salivary ducts, airway and the distal kidney nephron. The final regulation of Na⁺ reabsorption by the kidney occurs in the distal convoluted tubules, connecting tubules and collecting ducts where regulatory inputs alter Na⁺ absorption through ENaC. The reabsorption of Na⁺ is a major determinant of extracellular fluid volume and consequently thought to be involved in the regulation of blood pressure. However, silencing the expression of a-ENaC in the collecting duct did not impair sodium balance in a mouse model (Butterworth 2010). Abnormalities in ENaC function have been directly linked to several human disease states including Liddle syndrome, pseudohypoaldosteronism and cystic fibrosis, and may be implicated in salt-sensitive hypertension (Bhalla and Hallows 2008).

            ENaC comprises three homologous subunits (α, β and γ) that are each composed of two transmembrane domains, an extracellular loop and short N- and C-termini (Alvarez de la Rosa et al. 2000; Canessa et al. 1994; Canessa et al. 1993). ENaC is regulated by intracellular sodium concentration ([Na⁺]_i) in a process called feedback inhibition. An increase of [Na⁺]_i produces a reduction in the open probability of the channel (P_o) (Anantharam 2006; Knight et al. 2006) and regulates the number of channels at the surface membrane (Kellenberger et al. 1998; Kuche-Vihrog et al. 2008). Feedback inhibition depends on C-terminal PY motifs of ENaC (Kellenberger et al. 1998). The ENaC gain-of-function mutations in those motifs produce a hypertension phenotype in mammals, because of the increased Na⁺ absorption in distal tubules (Schild et al. 1996).

            ENaC belongs to the DEG/ENaC mechano-sensitive family of proteins and is considered to be sensitive to a variety of mechanical stimuli like stretch or hydrostatic pressure (Awayda et al. 1995; Ismailov et al. 1997; Ma et al. 2002; Palmer and Frindt 1996) and shear stress (Carattino et al. 2004; Carattino et al. 2005; Fronius et al. 2010; Fronius and Clauss 2008; Karpushev et al. 2010, Kashlan et al. 2018; Knoepp et al. 2017; Morimoto et al. 2005; Satlin et al. 2001). However, ENaC sensitivity to osmotic stimuli has been a controversial issue (Awayda and Subramanyam 1998; Böhmer and Wehner 2001; Bondarava et al. 2009; Ji et al. 1998, Rossier 1998; Schreiber et al. 2003). We already studied the relationship between mouse ENaC (mENaC) inhibition and mild hypotonic stimuli in Xenopus laevis oocytes (Galizia et al. 2013). However, the [Na⁺]_i effect on osmosensitivity of  ENaC has not been evaluated. Here we investigated the relationship between the inhibition of inward sodium currents during hypotonic stimuli and the [Na⁺]_i in X. laevis oocytes. Our results suggest that the inhibitory response of ENaC activity caused by hypotonic stimuli could be dependent on the [Na⁺]_i.

 

2.       MATERIALS AND METHODS

 

Microinjection of Xenopus oocytes

Adult female Xenopus laevis frogs were anesthetized with 0.3% tricaine (MS-222) and the oocytes were surgically removed from the abdominal incision. Oocytes were defolliculated using 1 mg/ml type IV collagenase for 30 minutes at 18ºC in agitation in OR-2 medium. Then the oocytes were placed in ND96 medium, containing (in mM) NaCl 96, KCl 2, CaCl₂ 2, MgCl₂ 1, HEPES 5 (pH 7.4) supplemented with 1 µg/ml gentamicin (Invitrogen). Oocytes were injected with a Drummond injector (Drummond, Broomall, PA) with 2 ng of α, β, and γ mENaC (total volume 50 nl). 24-48 h after ENaC injection we obtained steady currents in the range of microamperes (mA) so all the experiments were done in this period. In selected experiments, 4 ng of mENaC were injected.

 

Synthesis of ENaC cRNA

We synthesized complementary RNAs (cRNAs) for α, β, and γ mouse wild type ENaC subunits using the T3 mMessage Machine kit (Ambion, Austin, TX) (Assef et al. 2011; Galizia et al. 2013).

 

TEVC experiments

We used a standard two-electrode voltage clamp technique using a Warner Oocyte Clamp OC 725C (Warner Instruments, Hamden, CT) with a bath probe circuit (Assef et al. 2011; Galizia et al. 2013; Palma et al. 2016). We acquired data through Clampex 8.0 (Axon Instruments, Union City, CA) using a DigiData 1320A interface at 1 kHz and stored electronically on a PC hard disk. Micropipettes had resistances of 0.5-4 MΩ when filled with 3 M KCl. We clamped the bath with two chloride silver wires through 3 % agar bridges in 3 M KCl and positioned close to the oocyte. In the well with the oocyte, we estimated the bath-fluid resistance as the resistance between both electrodes (about 100-200 Ω). We perfused the oocyte chamber (1 ml/min) with a peristaltic pump (Dynamax RP-1; Rainin Instrument, Woburn, MA) and the solution ejected by a needle placed on top of the well containing the oocyte. Prior to the hyposmotic challenge oocytes were bathed in ND72 isotonic solution as a stabilization period for 10-15 minutes before the recordings. For the current-voltage (I-V) relationships, we applied a series of 500 ms voltage steps from –100 to +40 mV in 20 mV increments in isotonic and hypotonic condition (1.5 minutes of perfusion). The currents were measured after 400-500 ms. ENaC-mediated Na⁺ currents (INa_amil) were defined as the current difference measured in the absence versus the presence of 10 μM amiloride in the bath solution and were used for the determination of the reversal potential (E_revNa⁺).The amiloride-sensitive inward conductance (GNa_inward), was determined as the slope of the linear fit between –100 and –40mV. Hypotonic treatment consisted of perfusion with a hypotonic solution during 1.5 minutes. Relative inhibition of steady state INa_amil (I_r) during hypotonicity was calculated as (I_r) = 1 – (I_hypo/I_iso), where I_hypo is the INa_amil at –100 mV during hypotonicity and I_iso represents de INa_amil at –100 mV in isotonicity. Isotonic treatment during 1.5 minutes of perfusion did not produced any significative inhibition of INa_amil for any of the evaluated [Na⁺]_o condition.

 

Intracellular sodium [Na⁺]_i determination

ENaC-expressing oocytes incubated 24-48 h in ND10, ND20, ND40 and ND72 were evaluated when perfusing with ND72 solution. We determined the [Na⁺]_i using the E_revNa⁺ and solving the Nernst equation (Kashlan et al. 2011; Kellenberger et al. 1998; Kusche-Vihrog et al. 1998) as follows:

 

E_revNa⁺ =(RT/F)* [ln ([Na⁺]_o/[Na⁺]_i)]                                                                           (eq. 1)

With RT/F = 25.69 mV (at 25 °C):

[Na⁺]_i= [Na⁺]_o* [1/exp (E_revNa⁺/25.69)]                                                                        (eq. 2)

 

Inactivation of currents

 

            The data between the start (usually the first 20 ms after the initial voltage change were discarded) and the end of the –100 mV voltage episode were chosen for the exponential fit. The current in each episode was fitted to a mono exponential using the least squares method (Clampfit 8.0). Inactivation kinetics where analyzed by fit analysis to an exponential of the general form:

 

I =I_–100 * exp(– t / τ) + ΔI_V                                                                                                                                                                                      (eq. 3)

 

where t is time, τ the associated inactivation time constant, I_–100 the initial current and ΔI_V the magnitude of the exponential. To normalize for different expression levels between oocytes, we calculated ΔI_V/I_–100 (Awayda et al. 2000).

Solutions and osmolarities

 

Different incubations of ENaC injected oocytes were performed with varied sodium concentrations, maintaining osmolarity (210 ± 10 mOsM) with addition of different amounts of mannitol. ND10, ND20, ND40 and ND72 solutions were composed with 10, 20, 40, 72 mM of sodium chloride, respectively. Each mentioned solution was also composed of (in mM): 1 MgCl₂, 2 KCl, 1.8 CaCl₂ and 5 HEPES (pH 7.4). The concentration of mannitol in added in each incubation solution was respectively (in mM): 174, 154, 114 and 50 (210 ± 10 mOsM).

During the electrophysiological experiments two solutions were used: ND72 isotonic solution (osmolarity: 210 ± 10 mOsM) was prepared adding 50 mM of mannitol to the ND72 hypotonic solution (osmolarity: 160 ± 10 mOsM), thus maintaining the ion strength. Osmolarities were measured in a freeze point osmometer.

 

Statistical analysis

Data were expressed as mean ± S.E., n equals the number of independent experiments analyzed and N represents the number of frogs used in each experimental series. Statistical analysis was performed using t-test. Correlation analysis (Pearson) was performed with Sigma Plot. Differences were considered statistically significant when p<0.05.

 

 

3.       RESULTS

 

3.1. Extracellular sodium effect on the [Na⁺]_i and the conductance of mENaC-injected oocytes

 

            Our first step to study the effect of [Na⁺]_i on the hypotonic INa_amil response was to alter the [Na⁺]_i by performing incubations of mENaC-injected oocytes in ND solutions with different extracellular sodium concentrations for 24-48 h prior to measurements (see “Material and Methods”). Estimation of [Na⁺]_i in each oocyte was achieved using the ENaC reversal potential and solving Nernst equation (see “Materials and Methods”). Figure 1A shows the [Na⁺]_i dependence on the [Na⁺]_o in mENaC expressing oocytes. In each case, [Na⁺]_i is very close to [Na⁺]_o demonstrating that in mENaC expressing oocytes, these levels of sodium are near equilibrium.

            To evaluate the [Na⁺]_i influence on basal ENaC activity, the inward conductance (GNa_inward, see “Materials and Methods”) was calculated in oocytes expressing mENaC and plotted as a function of different [Na⁺]_o (Fig. 1B). Maximum GNa_inward is observed at ~20mM of [Na⁺]_o and GNa_inward decrement is observed at low (10 mM) and high (72 mM) [Na⁺]_o. This bell-shaped relationship between ENaC activity and [Na⁺]_o during incubation of mENaC-injected oocytes was previously reported by Kellenberger and colleagues, suggesting that mENaC activity can be controlled by the [Na⁺]_i (Kellenberger et al. 1998).

 

3.2. Role of the [Na⁺]_i on the hypotonic regulation of mENaC

 

            The next step was to evaluate the effect of hypotonic shock (–50 mOsM) on the INa_amil decrease (see “Materials and Methods”) in oocytes incubated at different [Na⁺]_o. After 24-48 h of incubation in ND72, ND40, ND20 and ND10 solutions, mENaC injected oocytes were challenged with hypotonic solution and INa_amil were measured. Figure 2A shows superimposed current traces in which the potential was held at 0 mV and jumped to values between –100 to +40 mV for 500 ms. Following a stabilization period of 10-15 minutes in the isotonic condition (ND72 isotonic: 210 mOsM) the oocytes were perfused with the hypotonic ND72 solution (160 mOsM) for 1.5 minutes. The rapid inhibitory effect on INa_amil during hypotonicity (1.5 minutes, –50 mOsM) is observed in 72 mM [Na⁺]_o (left panel). However the inhibitory effect of hypotonicity on INa_amil is not observed in the 20mM [Na⁺]_o (right panel).

 

Figure 2B shows the current-voltage relationships using the same [Na⁺]_o showed in Figure 1A in isotonic and hypotonic conditions. In ND72 incubated oocytes the GNa_inward is significantly reduced from 28.8 ± 5.4 µS to 14.4 ± 4 µS (n=13, N=4, p<0.05) in hypotonicity. On the other hand, the GNa_inward in the 20 mM condition only diminishes from 50.8 ± 8.9 µS to 36.5 ± 6.6 µS (n=13, N=4) in hypotonicity. Variations in [Na⁺]_i due to the hypotonic stimuli are negligible, because changes in E_revNa⁺ during the hypotonic shock were not observed at any [Na⁺]_i condition, as depicted in the I-V curves.

            In order to avoid the expression variability factor of ENaC between oocyte batches, the INa_amil relative inhibition (I_r) during hypotonicity was calculated in different [Na⁺]_i conditions. Figure 3A shows the I_r changes recorded at –100 mV in oocytes exposed to [Na⁺]_i values ranging between 10 mM and 72 mM in hypotonic condition. As expected from what we observed in experiments shown in Figure 1, I_r is smaller at 20 mM than at 72 mM (p<0.05). This behavior was also seen in the 40 mM medium (p<0.05), meanwhile at 10 mM no difference was observed.

To analyze the possibility that GNa_inward at these experiments affects ENaC inhibition by hypotonic stimuli, we plotted I_r versus the GNa_inward values obtained for each oocyte in the same experiment (Fig. 3B). A significant correlation with a negative slope arises from the cloud of points. This correlation strongly suggests that the inhibitory effect of hypotonic stimuli on ENaC is a function of GNa_inward (Pearson correlation coefficient = –0.45, p<0.05).Then we evaluated the role of the GNa_inward magnitude on the hypotonic inhibitory effect in oocytes incubated at the same [Na⁺]_o. For this, we compared the magnitude of I_r in oocytes with different basal GNa_inward induced by injection of two different masses of ENaC-cRNA (2 and 4 ng) at the same [Na⁺]_i. As expected, the greater mass of ENaC-cRNA provokes higher GNa_inward values (Fig. 3C). However, no changes in the I_r was observed in these two conditions (Fig. 3D).

Altogether, these results suggest a functional relationship between the osmotic sensitivity of mENaC and the [Na⁺]_i.

 

3.3. Effect of acute sodium load on the hypotonic regulation of ENaC by [Na⁺]_i

 

 

            Although our results may suggest that the regulation of ENaC by hypotonic stimuli is being modulated by the [Na⁺]_i, we wondered if the rapid changes seen in [Na⁺]_i might by itself affect ENaC osmosensitivity directly. To address this possibility, mENaC-injected oocytes incubated in 20 mM [Na⁺]_o for 24-48 h were exposed to 72 mM [Na⁺]_o prior to the experiment to increase the [Na⁺]_i. In this acute sodium load procedure experiment, the oocytes incubated in high [Na⁺]_o were clamped at –100 mV for 5 minutes to favor rapid sodium influx (Kellenberger et al. 1998). After this acute sodium load procedure, currents were evaluated in isotonic and hypotonic conditions as previously described. Figure 4A shows the [Na⁺]_i in oocytes previously incubated 24-48 h in ND20 and then incubated  with an acute loading of sodium in ND72 during 5 min to increase [Na⁺]_i to 65 ± 5 mM (n=7, N=3). With this experimental condition the hypotonic stimuli showed no inhibition (Fig. 4B). The I_r values obtained were not significantly different when compared with the control condition without the acute sodium load procedure (Fig. 4B). These results suggest that [Na⁺]_i per se does not exert a direct effect on ENaC currents.

 

3.4. Role of [Na⁺]_i on current inactivation kinetics during hypotonicity

 

            Hypotonic stimulus has been shown to elicit an inactivation of mENaC inward currents measured as a decrease in the time constant (τ) due to a P_o decrease (Galizia et al. 2013). Then we tested if the [Na⁺]_i can affect the inactivation rate of inward currents during hypotonicity. We analyzed the kinetics of INa_amil by fitting the values obtained with the highest hyperpolarized voltage used (–100 mV) to an exponential function. Then we calculated the inactivation τ and the magnitude of the exponential function (ΔI_V/I_–100 see “Materials and Methods”). These parameters represent the time course and amount of inactivation of currents, respectively, an approach previously used to quantify the rENaC activation to hyperpolarized stimuli (Awayda et al. 2000).

 

            When INa_amil was measured during the hyperpolarized pulse, in isotonic and hypotonic solutions for both 20 mM and 72 mM conditions, a slower inactivation current in the 20 mM [Na⁺]_i condition is evidenced (Fig. 5A). For isotonic and hypotonic stimuli, we analyzed the relationship between either τ (Fig. 5B) or the amount of inactivation (ΔI_V /I_–100) (Fig. 5C) for each [Na⁺]_i. As it was stated above (see “Materials and Methods”), the ΔI_V /I_–100 was calculated to normalize for possible expression-level differences among oocytes (Awayda et al. 2000). In isotonic media no differences in inactivation were observed at any [Na⁺]_i condition, however under hypotonic stimuli only the in the ND10 and the ND72 conditions both parameters of inactivation were affected (Fig 5B and C). Then for hypotonic stimuli, a bell-shaped (Fig 5B) and inverted bell-shaped relationship (Fig 5C) are observed in both figures. Taken together, this evidence suggests that the INa_amil decay during the hyperpolarized pulse (–100 mV) is the major contributor to the [Na⁺]_i dependent hypotonic inhibition.

 

4. DISCUSSION

           

            ENaC was demonstrated to participate in cell volume regulation and mechanosensation (Ji et al. 1998; Böhmer and Wehner 2000). Although mechanical stimuli as shear stress was extensively shown to regulate ENaC (Carattino et al. 2004; Fronious et al. 2008), the evidence regarding the regulation of ENaC by osmotic shocks remains controversial (Ji et al. 1998; Awayda and Subramanyam 1998; Schreiber et al. 2003, Galizia et al. 2013). In the last decades the regulation of the activity of ENaC by [Na⁺]_i was studied and it was proposed to be an important mechanism of regulation of ENaC activity (Kellenberger et al. 1998; Heidrich et al. 2015). Therefore, knowledge regarding the role of [Na⁺]_i on osmotic induced ENaC regulation may be crucial to understand ENaC osmosensitivity.

 

In this work we studied the influence of [Na⁺]_i on the modulation of ENaC by hypotonic stimuli using X. laevis oocytes expressing mENaC. We showed that varying [Na⁺]_i through incubation of the oocytes in solutions with different Na⁺ concentrations can affect the inhibition of ENaC during hypotonic stimuli. Our results indicate that the hypotonic-mediated inhibition of mENaC have a dependence on the different extracellular sodium incubation conditions. Although the effect of osmotic pressure on ENaC activity was previously evaluated in several systems and experimental conditions, the dependence of intracellular sodium on the hypotonicity regulated inhibition of ENaC was not previously evaluated (Galizia et al 2013; Ji et al. 1998). When ENaC injected oocytes were incubated in ND20 or ND40 extracellular solution, (i.e. intracellular sodium is around 20 or 40 mM, respectively) hypotonicity produces a reduced inhibitory response in comparison with the oocytes incubated at 72 mM. The results obtained here in high [Na⁺]_i conditions confirms previous findings by our group (Galizia et al. 2013). Interestingly, at the lowest [Na⁺]_i condition (10 mM), hypotonicity produced a inhibition similar to the observed at 72 mM. However the abolished response of hypotonicity in both 20 and 40 mM [Na⁺]_i conditions may explain the previous controversial findings by other groups (Awayda et al. 1998; Schreiber et al. 2003).On the other hand the acute [Na⁺]_i increase to values near 60 mM in oocytes previously incubated in a low sodium condition seems to be not enough to produce a recovery of the hypotonic mediated ENaC response. This suggests that the [Na⁺]_i at the moment of the experiment is not having a direct effect on ENaC activity. These findings may suggest the [Na⁺]_i is not having a direct effect on ENaC osmosensitivity. The observed correlation between GNa_inward and the inhibitory response to hypotonicity suggests a possible dependence of GNa_inward in the inhibition of ENaC by hypotonic stress in the different [Na⁺]_i conditions. However, in our hands the hypotonicity inhibition response seems to be independent of the increased GNa_inward when the injections of two different ENaC cRNA masses were evaluated. This evidence implies that [Na⁺]_i effect on GNa_inward, may affect the hypotonic response, but this effect seems not to be produced by the conductance per se. Previous data suggest that mechanical stretch, shear stress and hyposmotic stimuli can affect ENaC activity trough a modification in gating kinetics involving changes in P_o (Palmer and Frindt 1996; Carattino et al.2004; Fronius and Clauss et al. 2008; Galizia et al. 2013). Our data showing a [Na⁺]_i dependence on the inactivation of ENaC inward currents at hyperpolarized voltage pulses (see Fig. 5), suggests the idea of a voltage gated regulation during hypotonic stimuli. Although ENaC is a voltage insensitive channel, a number of mutations produce a masked voltage sensitivity and one of them is regulated by [Na⁺]_i (Kucher et al. 2011; Pochynyuk et al. 2009). Further analysis and experiments must be performed to understand the possibility of voltage regulation of ENaC osmosensitivity.

            The published reports about the role played by osmotic challenge of epithelial Na⁺ channels in X. laevis oocytes have generated conflicting results (Awayda and Subramanyam. 1998; Ji et al. 1998; Schreiber et al. 2003). These apparently controversial observations would be due to the different experimental approaches applied (Rossier 1998; Marunaka et al. 2014). The incidence of [Na⁺]_i on the osmosensitivity could be related to changes in physical membrane properties since [Na⁺]_i was shown to modify membrane rigidity through ENaC activity (Oberleithner 2007). Then, if [Na⁺]_i alters the rigidity of ENaC bearing membranes, the sensitivity of ENaC to an osmotic shock could also be affected. Although the mechanisms underlining the effects of [Na⁺]_i on the ENaC osmosensitivity remains unknown, the findings in this work could explain some of the prior controversial results.

 

Acknowledgments

 

            ENaC cDNAs were generously provided by Dr. M. Carattino (Pittsburgh, Pa, USA). The set for oocytes was a gift of Dr. C. Peracchia (Rochester, NY, USA).This research was funded by FONCYT grant number Prestamo BID PICT14 0357(to L.G).

 

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Rossier B (1998) Mechanosensitivity of the Epithelial Sodium Channel (ENaC): Controversy or Pseudocontroversy? J Gen Physiol 112: 95–96

 

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6. Figure Legends

 

Figure 1.[Na⁺]_i and GNa_inward in mENaC-injected oocytes incubated at different [Na⁺]_o. A. Four conditions of [Na⁺]_o during incubation were assessed: ND10, ND20, ND40 and ND72. These solutions were composed with 10, 20, 40 and 72 mM of sodium chloride, respectively (see “Material and Methods”). [Na⁺]_i was calculated from the Nernst equation (see eq.1 and eq.2) using the equilibrium potential of the I-V relationships of ENaC-expressing oocytes for each [Na⁺]_o condition. B. GNa_inward was calculated as described in “Materials and Methods”. A bell-shaped relationship is observed with a maximal GNa_inward at 20mM [Na⁺]_i and decrements at low (10 mM) and high (72 mM) [Na⁺]_i. 10-13 oocytes corresponding to 4 batches per condition are shown. Asterisk indicates differences in GNa_inward. Results are expressed as mean ± SE.

 

Figure 2.Role of [Na⁺]_I on the inhibition of INa_amil by hypotonic stimuli. A. Representative recordings of mENaC-expressing oocytes incubated in high (left, ND72) and low (right, ND20) sodium concentrations before (isotonic) and after the hypotonic shock (–50 mOsM, 1.5 min), after subtracting the amiloride sensitive component B. Current-voltage (I-V) relationship of amiloride-sensitive steady state whole cell currents (INa_amil) in hypotonic treated oocytes. INa_amil from mENaC-expressing oocytes where recorded before (filled circles) and after (open circles) 1.5 minutes perfusion with the hypotonic solution in both conditions. The inhibitory effect induced by hypotonicity on INa_amil is smaller in the 20 mM condition. Results are expressed as mean ± SE.

 

Figure 3.Relative inhibition of INa_amil (I_r) during hypotonicity and role of GNa_inward. A. I_r was calculated in mENaC-injected oocytes pre-incubated in different extracellular sodium conditions. I_r decreased at 20 mM and 40 mM, but not at ND10 when compared with the ND72 condition (p< 0.05, t-test, 10-13 oocytes and 4 batches per condition are shown). B. A strong negative correlation (Pearson correlation coefficient = –0.45, p<0.05, n=41) is observed between GNa_inward at the different [Na⁺]_i and the obtained hypotonicity response (I_r). C. Different conductances (GNa_inward) can be induced by injecting different masses of cRNA-ENaC. D. The relative inhibition of inward currents (I_r) was calculated during hypotonicity in oocytes injected with two mENaC-cRNA concentrations. Oocytes were pre-incubated with the same extracellular sodium concentrations. When 4 ng of mENaC-cRNA were injected, GNa_inward was increased 3-fold (C). However, I_r at those GNa_inward conditions remains constant (D) (data from 12-13 oocytes and 4 batches per condition are shown). Asterisk indicates differences in I_r or GNa_inward (p<0.05, t-test).

 

Figure 4.Acute sodium load does not affect the inhibition of ENaC inward sodium currents by hypotonic stimuli. Oocytes were incubated in a low sodium medium (ND20) during the expression phase to maintain [Na⁺]_i. Before the experiment, perfusion of the oocytes with a solution containing high sodium concentration (ND72) was started, and oocytes were voltage-clamped to –100 mV to favor rapid sodium influx (Kellenberger et al. 1998). A. Changes in [Na⁺]_i measured from the reversal potential of INa_amil during the acute sodium load [Na⁺]_i increased rapidly over the first 5 min of perfusion with ND72. Asterisk indicates differences in [Na⁺]_i (p<0.05, t-test).B. The relative inhibition during hypotonic stimuli in mENaC-injected oocytes pre-incubated in control (filled bar) and acute sodium load conditions (empty bar) shows no difference (n =7, N =3). Results are expressed as mean ± SE.

 

Figure 5.Inactivation of INa_amil by hypotonic stimuli. A. Representative pulses of INa_amil in isotonic and hypotonic conditions. The inactivation of INa_amil during hypotonic stimuli pre-incubated in different [Na⁺]_o was calculated by fitting the curves to an exponential function (see Materials and Methods). There is a slower inactivation current at 20 mM of [Na⁺]_i. B. The time constant of inactivation (τ) of INa_amil in hypotonic stimuli at –100 mV depends on the [Na⁺]_i. At 10 mM and 72 mM τ decreases in hypotonicity. C. The amount of inhibition at a hyperpolarized pulse (–100 mV) during hypotonicity (ΔI_V/I_–100, see Materials and Methods) also depends on the [Na⁺]_i. At 10 mM and 72 mM, ΔI_V/I_–100 increases in hypotonicity. Asterisk indicates differences with ND72 (p< 0.05, t-test). 10 to 13 oocytes from 4 batches per condition are shown. Results are expressed as mean ± SE.