Evidence for ammonium conductance in a mouse thick ascending limb cell line

Abstract In this study, we examined an ammonium conductance in the mouse thick ascending limb cell line ST‐1. Whole cell patch clamp was performed to measure currents evoked by NH 4Cl in the presence of BaCl2, tetraethylammonium, and BAPTA. Application of 20 mmol/L NH 4Cl induced an inward current (−272 ± 79 pA, n = 9). In current‐voltage (I–V) relationships, NH 4Cl application caused the I–V curve to shift down in an inward direction. The difference in current before and after NH 4Cl application, which corresponds to the current evoked by NH 4Cl, was progressively larger at more negative potentials. The reversal potential for NH4Cl was +15 mV, higher than the equilibrium potential for chloride, indicating that the current should be due to NH 4 +. We then injected ST‐1 poly(A) RNA into Xenopus oocytes and performed two‐electrode voltage clamp. NH 4Cl application in the presence of BaCl2 caused the I–V curve to be steeper. The NH 4 + current was retained at pH 6.4, where endogenous oocyte current was abolished. The NH 4 + current was unaffected by 10 μmol/L amiloride but abolished after incubation in Na+‐free media. These results demonstrate that the renal cell line ST‐1 produces an NH 4 + conductance.


Introduction
NH þ 4 is a key buffer component that regulates blood pH. In essence, the kidneys excrete NH þ 4 to urine as they produce HCO À 3 , and the mechanism by which NH þ 4 excretion results in net acid excretion involves a series of sophisticated NH þ 4 transport processes in different parts of the nephron (Weiner and Verlander 2013;Hamm et al. 2015). One of the nephron segments that play key roles in NH þ 4 excretion is the thick ascending limb (TAL) (Mount 2014). NH þ 4 transport in the TAL involves the Na/K/2Cl cotransporter NKCC2 (Good et al. 1984;Kinne et al. 1986), K/NH 4 exchange and NH þ 4 conductance (Amlal et al. 1994;Attmane-Elakeb et al. 2001) in the luminal membrane of the tubule, and the Na/H exchanger NHE4 (Bourgeois et al. 2010) in the basolateral membrane. The basolateral NH þ 4 transport is also mediated by a dissociation of intracellular NH þ 4 into NH 3 and H + and subsequent NH 3 exit to the interstitium.
In the luminal membrane of the TAL, NKCC2 is the major fraction of the active NH þ 4 flux. Nonetheless, in vitro studies reveal that K/NH 4 exchange and NH þ 4 conductance can contribute to the NH þ 4 transport by 35-50% (Amlal et al. 1994;Attmane-Elakeb et al. 2001). K/NH 4 exchange is barium-and verapamil-sensitive, whereas NH þ 4 conductance is barium-insensitive and amiloride-sensitive (Amlal et al. 1994). The two pathways exhibit biophysical and pharmacological characteristics that distinguish them from other NH þ 4 -transporting proteins. Despite such physiological and functional significance, our understanding of these pathways is limited because their molecular entities are presently unknown.
In this study, we examined an NH þ 4 conductance in the mouse TAL cell line ST-1. This cell line is nonpolarized and exhibits many features characteristic of TAL cells (Kone et al. 1995;Kone and Higham 1999;Lee et al. 2010). We performed whole cell patch clamp of the cells to identify the NH þ 4 conductance and determine basic electrophysiological properties such as the amount of current, direction, currentvoltage relationship, and reversal potential. We then isolated ST-1 poly(A) RNA and injected it into Xenopus oocytes and performed two-electrode voltage clamp in an effort to identify a protein conducting NH þ 4 . While our search for the protein is in progress, here we report that the NH þ 4 conductance in ST-1 cells is not identical to the previously reported Cl -dependent NH þ 4 conductance in the TAL of the nephron.

Ethical approval
All experiments in this study were conducted under the National Institutes of Health guidelines for research, and experimental protocols were approved by the Institutional Animal Care and Use Committee at Emory University.

Cell culture
ST-1 is a cell line derived from mouse medullary TAL tubules, developed by Bruce Kone (Kone et al. 1995;Kone and Higham 1999). Cell authentication is based on the report (Haas and Hebert 1992) on the expression of bumetanide-sensitive proteins and our previous report (Lee et al. 2010) demonstrating the expression of the electroneutral Na/HCO 3 transporter NBCn1 and Cl/HCO 3 exchanger AE2 in this cell line by immunoblot. Cells were cultured in Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin and 50 lg/mL streptomycin in a 5% CO 2 air equilibrated 37°C incubator. For patch clamp recording, cells were seeded on poly-D-lysine-coated coverslips at a density of 8 9 10 4 cells per well in a 12-well plate. Recordings were done 2 days later.

Whole cell patch clamp of ST-1 cells
Whole cell recording was performed using the protocol by Hayashi et al. (1992)  Cl were recorded in the absence and then presence of 1 lmol/L amiloride (Sigma-Aldrich; Cat#: A7410). I-V relationship was determined by a staircase voltage command between À80 to +15 mV (170 msec duration). Currents were recorded using pClamp 8.0 (Molecular Devices) and signals were low-pass filtered (3 db at 2 kHz, 8-pole Bessel filter). Experiments were performed at room temperature.

Two-electrode voltage clamp of oocytes
ST-1 cells grown in 100 mm plates were collected and lysed for poly(A) RNA isolation using RNeasy Mini kit (Qiagen, Germantown). Xenopus laevis oocytes at stages V and VI were purchased from Ecocyte Bioscience (Austin). Poly(A) RNA was injected into oocytes (2.7 ng per oocyte in 46 nl) and controls were water only. Oocytes were maintained for 3 days at 18°C before use. A RC-24N recording chamber (Warner Instrument, Hamden) was filled with perfusion solution containing (in mmol/L) 20 LiCl, 80 NaCl, 3.8 BaCl 2 , and 5 HEPES, pH 7.4. For acidic solution, the pH was adjusted to 6.4. An oocyte was placed in the chamber and impaled with two glass electrodes filled with 3 mol/L KCl (a tip resistance of 0.5-2 mol/LΩ). The oocyte was clamped at -60 mV using a OC-725C voltage-clamp amplifier (Warner Instrument). NH 4 Cl solutions were made by replacing LiCl at equimolar concentrations. Na + -free solutions were made by replacing Na + with N-methyl-D-glucamin (NMDG). For drug sensitivity experiments, 10 lmol/L amiloride and 200 lmol/L bumetanide (Sigma-Aldrich; Cat #: B3023) were used. The voltage command was from -140 to +40 mV with 20 mV increments (100 msec duration). The voltage command after NH 4 Cl application was made when the current reached steady state (~4 min) (Lee and Choi 2011). Voltage signals were sampled by a Digidata 1322A (Molecular Devices) and data were acquired using pClamp 8.0.

Statistical analysis
Data were reported as mean AE standard error. The level of significance was determined using (1) paired, twotailed Student t-test for comparison of slopes before and after NH þ 4 application to oocytes or ST-cells, and comparison of the currents before and after NH 4 Cl (2) unpaired, two-tailed Student t-test for comparison of G NH4 between RNA-injected versus water-injected oocytes; and (3) two-way ANOVA with Bonferroni post hoc test for comparison of slopes in the presence of bumetanide and in Na + -free solutions between different groups of oocytes. The P value of less than 0.05 was considered significant. Analysis was made using Microsoft Office Excel add-in program Analysis ToolPak (Redmond, WA).

Results
NH þ 4 conductance in ST-1 cells We performed patch clamp recording of ST-1 cells in a whole cell configuration to assess NH þ 4 conductance.
Recordings were performed in the presence of 1 mmol/ L BaCl 2 and 10 mmol/L TEA to block K channels, and 5 mmol/L BAPTA to block intracellular Ca 2+ increase. Figure 1A shows an example of the inward current evoked by 20 mmol/L NH 4 Cl at the holding potential of À70 mV. As in many cells, the current was decayed slowly but we also frequently observed steady-state currents after reaching a peak. Figure 1B shows the currents measured before and after NH 4 Cl application. The mean difference between the two values, corresponding the current evoked by NH 4 Cl, was À272 AE 79 pA (P < 0.05; n = 9). In I-V relationships (Fig. 1C), NH 4 Cl application caused the I-V curve to shift down in an inward direction. The difference in the two curves, corresponding to the currents evoked by NH 4 Cl at different voltages, was progressively larger at more negative potentials. The reversal potential for NH 4 Cl was +15 mV, higher than the equilibrium potential for chloride (À4 mV) estimated by chloride concentrations in the patch pipette and bath solutions. Thus, the inward current evoked by NH 4 Cl is not due to Cl -, but to NH þ 4 . The NH þ 4 conductance (G NH4 ) determined by the difference in the slopes of the two I-V curves was 4.4 nS (Fig. 1D). In other experiments, we determined the effect of amiloride on the currents (Fig. 1E). Measured at the holding potential of À70 mV, the currents were unaffected by 1 lmol/L amiloride (P > 0.05; n = 6).

G NH4 induced by ST-1 proteins
To express ST-1 proteins in Xenopus oocytes, we isolated poly(A) RNA from the cells and injected it into oocytes. Figure 2A shows I-V relationships of water-injected control oocytes, obtained before and after application of 20 mmol/L NH 4 Cl in the presence of 3.8 mmol/L BaCl 2 . NH 4 Cl caused the I-V curve to shift down in an inward direction. This shift is due to endogenous oocyte conductance (Lee and Choi 2011). A slight increase in an outward current was observed, probably due to LiCl that depolarizes oocyte membranes. In oocytes injected with  (Fig. 2B), the basal current before NH 4 Cl application was higher, due to Na + as describe below. NH 4 Cl caused the I-V curve to shift down with a steeper slope. At À60 mV, the inward current in these oocytes was À780 AE 164 nA, significantly higher than À240 AE 12 nA in controls. The reversal potential (the voltage where the two I-V curves intersect) was similar between control oocytes and RNA-injected oocytes, indicating that the currents are likely produced by nonselective cation channels. Figure 2C shows the comparison of the slopes determined near the reversal potential. RNAinjected oocytes had a more increased slope in response to NH 4 Cl (P < 0.05 for both; n = 7 for RNA-injected oocytes and 5 for water-injected controls). Thus, the G NH4 in RNA-injected oocytes was significantly larger than that of controls (P < 0.05; Fig. 2D). In other experiments, we examined the effect of amiloride on the slope induced by NH 4 Cl (Fig. 2E). We found no significant change by 10 lmol/L amiloride (P > 0.05; n = 5 for each).

Retention of ST-1 G NH4 at acidic pH
We performed I-V recordings in solutions with pH 6.4 to determine whether endogenous oocyte conductance was responsible for increased G NH4 in RNA-injected oocytes. Endogenous oocyte NH þ 4 transport is known to be inhibited at low pH (Nakhoul et al. 2010). We found that while the G NH4 in control oocytes was abolished at pH 6.4, the one in RNA-injected oocytes was still induced under the same condition (P < 0.05; Fig. 3). Thus, the G NH4 in RNA-injected oocytes is mainly induced by heterologously expressed ST-1 proteins.

Inhibition of G NH4 by incubation in Na + -free solutions
To test whether the G NH4 in RNA-injected oocytes is affected by Na + , we incubated oocytes in Na + -free solutions (NMDG replaced Na + ) and determined I-V relationships. The incubation time was at least 3 h to ensure that intracellular Na + is substantially low to minimize Na + efflux. Figure 4A shows the I-V relationships under Na + -free conditions. NH 4 Cl application caused the I-V curve of control oocytes to inwardly shift down, indicating negligible effect of Na + removal on endogenous conductance. In contrast, Na + removal induced two changes in RNA-injected oocytes. First, the I-V curve before NH 4 Cl application was similar to the control curve. Second, the shift in the curve after NH 4 Cl application was smaller than the control. These changes were evident when the slopes of the curves were compared (Fig. 4B). Compared to controls, RNAinjected oocytes had a similar slope of the basal current (P > 0.05; n = 7 for RNA-injected oocytes and 6 for controls) but a small slope of the NH þ 4 current (P < 0.05). This resulted in a smaller G NH4 in RNAinjected oocytes (Fig. 4C). To test whether the 'belowcontrol' decrease in G NH4 is associated with NKCC2, we treated oocytes with 200 lmol/L bumetanide under Na + -free conditions. Figure 4D shows an example of I-V relationships after treating with bumetanide. The I-V curves after NH 4 Cl application were nearly superimposed between controls and RNA-injected oocytes, indicating that bumetanide unleashed the excessive inhibition of the G NH4 by Na + removal. The two groups of oocytes had similar slopes and G NH4 (P < 0.05; n = 6 for RNA-injected oocytes and 7 for controls), as  Figure 4E and F. We also used 500 lmol/L of bumetanide and found the same effects (data not shown).

Discussion
The aim of this study was to obtain the basic electrophysiological properties of the NH þ 4 conductance in the TAL cell line ST-1 for future study of its molecular identification. Using a homogeneous cell line and its protein expression in a relatively simple heterologous oocyte system, we identified an NH þ 4 conductance and obtained information on the direction of the current flow, amounts of currents produced, I-V relationships, and ion dependence. An interesting finding is that this conductance is not inhibited by amiloride and appears to be dependent upon Na + . This finding is significant as it provides evidence that the NH þ 4 conductance in ST-1 is different from the previously reported Cl --dependent NH þ 4 conductance in the TAL of a nephron. Amlal et al. (1994) have reported the NH þ 4 conductance in the isolated rat medullary TAL by monitoring membrane depolarization of the tubules using DiSC 3 (5). While this approach determines whether there is a current flowing across the membrane, it does not measure actual membrane conductance. Current is proportional to conductance at a constant voltage, and a voltage or current clamp should be done to correctly measure a membrane conductance. In our study, we directly measured the current evoked by NH 4 Cl. The current was inwardly directed with a positive reversal potential and a positive G NH4 was observed. These data are consistent with an inward movement of positively charged NH þ 4 ions. This inward current is unlikely mediated by Cl -. Given the Clconcentrations in the patch pipette and in the bath, the equilibrium potential for Clis estimated to be slightly negative. The voltage-clamp experiments of oocytes shows that the NH þ 4 conductance is produced in the presence of barium. Patch clamp of ST-1 cells also shows NH þ 4 conductance in the presence of barium and TEA, consistent with the report that NH þ 4 is poorly transported by K channels in the TAL (Attmane-Elakeb et al. 2001).
We found that the G NH4 was inhibited after incubation in Na + -free solutions (Fig. 4). This Na + -dependent inhibition is unexpected because an NH þ 4 current is considered to occur via K + channels (or transporters) by replacing a K binding site. One explanation is that Na + -free incubation has lowered intracellular pH by reversing the Na/H exchangers and then inhibited the G NH4 . However, this is unlikely to happen because the G NH4 can be produced at acidic pH as shown in Figure 3. Another explanation is that Na + -free solutions has inhibited Na + channels or Na + channel-like proteins, which may induce NH þ 4 currents. NH þ 4 affects activities of epithelial sodium channel ENaC (Nakhoul et al. 2001) and gates acid-sensing ion channel ASICs (Pidoplichko and Dani 2006). Nonetheless, we do not think that ENaC and ASICs are responsible for the G NH4 in ST-1 because these two proteins are very sensitive to amiloride (Benos 1982;Wemmie et al. 2006). We found no amiloride sensitivity of the G NH4 . The NH þ 4 conductance in the TAL is sensitive to amiloride given that 1 lmol/L amiloride completely abolishes the NH 4 Clinduced membrane depolarization in the isolated TAL tubules (Amlal et al. 1994). The TAL does not express ENaC although we note that ENaC antibodies detect signals in the luminal side of rat TAL (Brown et al. 1989). Taken together, we think that the molecule responsible for NH þ 4 conductance in ST-1 is a novel protein that is not inhibited by amiloride and has sensitivity to Na + , probably to intracellular Na + . ST-1 RNA-injected oocytes do not regain G NH4 after incubation in Na + -free solutions (S. Lee, unpublished observation), implying that the intracellular Na + levels are critical for G NH4 .
What would be a potential role of the G NH4 in renal ammonium excretion? We think that the conductance would contribute to renal adoptive process in acid-base disorders. For example, in rats and humans, K + depletion is associated with increased urinary NH þ 4 production and excretion, ultimately developing metabolic alkalosis (Jones et al. 1982;Abu Hossain et al. 2011). While this development is probably due to increased ammoniagenesis in the proximal tubules, K + depletion downregulates NKCC2 in the TAL (Amlal et al. 1998). Thus, it is possible that other mechanisms such as NH þ 4 conductance are upregulated during K + depletion. In addition, the G NH4 may serve as a fine-tuning regulator of NH þ 4 transport in the TAL, where luminal NH þ 4 transport is mainly mediated by electroneutral NKCC2. The regulation of G NH4 by Na + might be a novel mechanism that links NKCC2 to NH þ 4 absorption and subsequent excretion.
In conclusion, our findings are interesting and provide a foundation for future studies of electrophysiological and pharmacological properties of the NH þ 4 conductance. Those data will subsequently lead to obtaining molecular information on an ammonium channel.