Inhibition of ROMK Channels by Low Extracellular K and Oxidative Stress

Am J Physiol Renal Physiol 305: F208–F215, 2013. First published May 15, 2013; doi:10.1152/ajprenal.00185.2013.

Inhibition of ROMK channels by low extracellular Kϩ and oxidative stress

Gustavo Frindt,1 Hui Li,2 Henry Sackin,2 and Lawrence G. Palmer1 1Department of Physiology and Biophysics, Weill-Cornell Medical College, New York, New York; and 2Department of Physiology and Biophysics, The Chicago Medical School, Rosalind Franklin University, North Chicago, Illinois Submitted 2 April 2013; accepted in ﬁnal form 8 May 2013

Frindt G, Li H, Sackin H, Palmer LG. Inhibition of ROMK may be essential for preventing Kϩ secretion and minimizing channels by low extracellular Kϩ and oxidative stress. Am J Physiol K losses. Renal Physiol 305: F208–F215, 2013. First published May 15, 2013; Measurements of ROMK activity in heterologous expression doi:10.1152/ajprenal.00185.2013.—We tested the hypothesis that low systems indicate that the channels are sensitive to changes in luminal Kϩ inhibits the activity of ROMK channels in the rat cortical ϩ ϩ ϩ the extracellular K concentration ([K ]o); decreases in [K ]o collecting duct. Whole-cell voltage-clamp measurements of the com-

downregulate the channels (7, 28, 29, 31). One aspect of this Downloaded from ponent of outward Kϩ current inhibited by the bee toxin Tertiapin-Q ϩ response is a shift in the dependence of channel activity on (ISK) showed that reducing the bath concentration ([K ]o)to1mM intracellular pH, with low [Kϩ] moving the titration curve for resulted in a decline of current over 2 min compared with that o ϩ inhibition of the channels toward a higher, more physiological observed at 10 mM [K ]o. However, maintaining tubules in 1 mM ϩ pH range. Such a mechanism could be relevant in vivo, since [K ]o without establishing whole-cell clamp conditions did not affect ϩ ϩ under conditions of K deprivation the luminal K concentra- ISK. The [K ]o-dependent decline was not prevented by increasing tion in distal nephron segments can fall to 1–2 mM (23; see cytoplasmic-side pH or by inhibition of phosphatase activity. It was, ϩ ϩ however, abolished by the inclusion of 0.5 mM DTT in the pipette Fig. 1). However, the effect of reducing [K ]o on K channels http://ajprenal.physiology.org/ solution to prevent oxidation of the intracellular environment. Con- in renal tubules has not been examined. ϩ versely, treatment of intact tubules with the oxidant H2O2 (100 ␮M) In this study, we test the hypothesis that lowering [K ]o in ϩ ϩ decreased ISK ina[K ]o-dependent manner. Treatment of the tubules K -secreting cells can directly reduce ROMK channel activity. with the phospholipase C inhibitor U73122 prevented the effect of We provide evidence for such a reduction, which occurs most ϩ low [K ]o, suggesting the involvement of this enzyme in the process. particularly under conditions of oxidative stress. We examined these effects further using Xenopus oocytes expressing ROMK2 channels. A 50-min exposure to the permeant oxidizing METHODS agent tert-butyl hydroperoxide (t-BHP; 500 ␮M) did not affect out- ϩ ϩ ward K currents with [K ]o ϭ 10 mM but reduced currents by 50% Animals. All procedures using animals were approved by the ϩ ϩ with [K ]o ϭ 1 mM and by 75% with [K ]o ϭ 0.1 mM. Pretreatment Institutional Animal Care and Use Committee of Weill-Cornell Med-

of the oocytes with U73122 prevented the effects of t-BHP. Under ical College or the Chicago Medical College. Sprague-Dawley rats by Henry Sackin on July 30, 2013 conditions of low dietary K intake, Kϩ secretion by distal nephron (150–200 g) of either gender (Charles River Laboratories, Kingston, ϩ NY) raised free of viral infections were used for all experiments. segments may be suppressed by a combination of low luminal [K ]o and oxidative stress. Animals were fed a conventional rodent diet (Purina). Stage V–VI oocytes were obtained by partial ovariectomy of female Xenopus ROMK channels; Kϩ secretion; oxidation; phospholipase C; U73122 laevis (NASCO, Ft. Atkinson, WI), while animals were anesthetized with tricaine methanesulfonate (1.5 g/l, adjusted to pH 7.0). Oocytes were defolliculated by incubation (on a Vari-Mix rocker) in Ca-free ϩ MODULATION OF THE RATE OF K secretion by the cortical collecting modified Barth’s solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, duct (CCD) and related nephron segments is important for match- and 5 mM HEPES, adjusted to pH 7.5 with NaOH) containing 2 ing renal K excretion with dietary K intake. When K intake is mg/ml collagenase type IA (cat no. C9891; Sigma Chemical, St. high, several adaptations occur that stimulate Kϩ secretion includ- Louis, MO) for 90 min and (if necessary) another 90 min in a fresh enzyme solution at 23°C. Oocytes were injected with 5–10 ng of ing increased activity of apical ROMK channels (13, 24, 37, 39), ϫ ϩ cRNA and incubated at 19°C in 2 diluted Leibovitz medium (Life activation of epithelial Na channels (12, 24), upregulation of Technologies, Grand Island, NY) for 1–3 days before measurements basolateral Na-K-ATPase (24, 32), and suppression of the up- were made. stream NaCl cotransporter NCC (11, 16, 35). The last response Electrophysiology. Measurement of whole-cell Kϩ currents in will increase Naϩ delivery to the more distal segments, reducing principal cells of the CCD followed procedures described previously NaCl reabsorption and enhancing Naϩ/Kϩ exchange. (10, 13). Split-open tubules were superfused with solutions pre- The mechanisms involved in limiting Kϩ secretion when warmed to 37°C containing the following (in mM): 5 NaCl, 130 Na methanesulfonate, 10 K methanesulfonate, 2 Ca methanesulfonate, 1 dietary intake is low may be different from those that upregu- ϩ MgCl2, 2 glucose, and 10 HEPES adjusted to pH 7.4 with NaOH. late K transport. ROMK activity was reported to be reduced ϩ [K ]o was reduced to 1 mM by substitution of K methanesulfonate in some (1, 13) but not other (25, 39) studies. The downregu- with Na methanesulfonate. Methanesulfonate was used to minimize lation is thought to be mediated, at least in part, by activation whole-cell anion currents. of the tyrosine kinase c-src (4, 22, 39). ROMK protein expres- For measurements of Kϩ currents through ROMK channels, the sion is also reduced by low K intake (11, 36). However, these patch-clamp pipettes were filled with solutions containing the follow- decreases are relatively modest, suggesting that other events ing (in mM): 7 KCl, 123 aspartic acid, 5 EGTA, and 10 HEPES, with the pH adjusted to 7.4 with KOH. The total concentration of Kϩ was ϳ145 mM. Tertiapin-Q (TPNQ; Sigma-Aldrich) was dissolved in ␮ Address for reprint requests and other correspondence: L. G. Palmer, Dept. H2O at a concentration of 100 M and diluted into the bath solution of Physiology and Biophysics, Weill-Cornell Medical College, 1300 York to a final concentration of 100 nM. Ba acetate was added to the bath Ave. New York, NY 10065 (e-mail: [email protected]). solution to a final concentration of 5 mM.

F208 1931-857X/13 Copyright © 2013 the American Physiological Society http://www.ajprenal.org LUMINAL Kϩ AND ROMK CHANNELS IN CCD F209 Pipettes were pulled from hematocrit tubing, coated with Sylgard, A 10 K and ﬁre polished with a microforge. Pipette resistances ranged from 2 1.4 ⍀ to5M . Currents were measured with a List EPC-7 ampliﬁer (Heka TPNQ Elektronik, Lambrecht, Germany). Voltages were controlled and cur- 1.2 rents were recorded using pClamp software and a Digidata 1320 1.0 interface (Molecular Devices, Sunnyvale, CA). Xenopus oocytes. Whole-cell currents and conductances were mea- 0.8

sured in intact oocytes using a two-electrode voltage clamp (Model nA 0.6 CA-1; Dagan, Minneapolis, MN) with 16 command pulses of 30-ms duration between Ϫ110 and ϩ50 mV, centered around the resting 0.4 potential. Upward deﬂections from the zero current line (dashed) denote outward current. 0.2 Oocytes expressing Kir1.1b (ROMK2) were bathed in permeant 0.0 acetate buffers to control their internal pH as previously described (5). 0 20 40 60 80 100 120 140 Variations in external (bath) [K] at constant ionic strength were s ϩ ϩ accomplished with 0.1 mM KCl 50 mM NaCl, 1 mM KCl 49 Downloaded from mM NaCl, or 10 mM KCl ϩ 40 mM NaCl, with the remainder of 10 K 1 K 1.2 bathing solution being 50 mM Na-acetate, 1 mM MgCl2,2mM CaCl2, and 5 mM HEPES. The pH of the bath was adjusted to control 1.0 internal oocyte pH at 7.8 with the acetate buffer system. A membrane-permeant analog of hydrogen peroxide [tert-butyl 0.8 hydroperoxide (t-BHP)] was obtained from Sigma Chemical (no. 0.6

19997) and used at a concentration of 500 ␮M. The PLC inhibitor nA http://ajprenal.physiology.org/ U73122 (cat. no. 662035) and the related compound U73343, lacking 0.4 PLC activity (cat. no. 662041), were both obtained from EMD TPNQ Millipore (Billerica, MA). 0.2 Statistics. Statistical differences were evaluated using the two-tailed 0.0 Ͻ Student’s t-test. The threshold for statistical significance was P 0.05. 0 20 40 60 80 100 120 140 RESULTS s ϩ We chose to investigate the influence of [K ]o over the B form whole-cell range of 1 to 10 mM based on the micropuncture data from rat + 1.2 in 10 mM [K ] kidney of Malnic et al. (23) obtained from superficial distal o + 1 mM or 10 mM [K ]o

convoluted tubules, redrawn in Fig. 1. These measurements by Henry Sackin on July 30, 2013 ϩ for 2 min indicate that with a low-K diet, [K ] in the luminal ﬂuid is 1 ! maintained Ͻ2 mM along the micropuncture-accessible distal add TPNQ tubule while concentrations rise to 10–15 mM in the second ROMK activity half of the tubules, with a normal K intake. The effect of 0.8 ϩ reducing [K ]o during a whole-cell clamp recording is illus- * trated in Fig. 2A. Whole-cell clamp conditions were established in two separate principal cells, both in the presence of 10 mM 0.6 ϩ ϩ 10K->10K [K ]o. The holding potential was zero mV, and the K concentration in the pipette, and by extension that in the cell (140 10K->1K mM), was much larger than that of the bath, establishing a 0.4 driving force for outward current carried by Kϩ.Inthetop normalized current ϩ trace,10mM[K ]o were maintained for ϳ2 min as a time control. At the end of this period, TPNQ (10Ϫ7M) was added 0.2

25 0 initial final TPNQ ISK control diet ϩ ϩ 20 Fig. 2. Effect of decreasing extracellular K concentration ([K ]o) on ROMK low-K diet currents in the rat cortical collecting duct (CCD). A: whole-cell recording 15 showing outward currents at a holding potential of 0 mV. Whole-cell clamps ϩ were formed in the presence of 10 mM [K ]o. Currents were stable in the presence of 10 mM [Kϩ] but declined with 1 mM [Kϩ] . At the end of the 10 o o recording Tertiapin-Q (TPNQ; 100 nM) was added to the medium to measure the ROMK-speciﬁc currents. B: summary of 8 similar pairs of experiments. tubular fluid [K] 5 Data represent means Ϯ SE. *P Ͻ 0.05, signiﬁcant difference.

20 40 60 80 ϩ to the bath and outward K current (ISK), the current passing percent distal tubule through ROMK channels, was measured as that inhibited by 2ϩ Fig. 1. Data from Malnic et al. (23) are replotted to show that with a low-K diet the toxin. The residual current was blocked by Ba (13) and ϩ luminal Kϩ concentration ([Kϩ]) is Յ 2 mM. presumably is carried by basolateral K channels. In the

AJP-Renal Physiol • doi:10.1152/ajprenal.00185.2013 • www.ajprenal.org F210 LUMINAL Kϩ AND ROMK CHANNELS IN CCD bottom trace, after a period of 10–15 s to establish baseline, the ϩ bath solution was exchanged for one containing 1 mM [K ]o. A 1 K 10 K Under these conditions, the outward current fell progressively 600 over a 2-min period, despite an increased driving force. After TPNQ this period there was only a small response to TPNQ. Figure 500 2B shows a summary of this and similar experiments. The data 400 indicate a strong inhibition of ISK in response to lowering ϩ [K ]o. In both cases TPNQ abolished a large fraction of the ϩ ϩ 300 total outward K current. This suggests that the basolateral K pA currents, assumed to be TPNQ-insensitive, are small. This is 200 due in part to the rapid rundown of these currents as described previously (13). It is also possible that the basolateral Kϩ 100 ϩ channels are affected by low [K ]o and contribute to the 0 decline in currents observed in Fig. 2A. We have not explored 0 20 40 60 80 100 120 140 this systematically. s Downloaded from Figure 3 shows the converse experiment. Here the whole- ϩ 1 K cell recordings were established in the presence of 1 mM [K ]o and the concentration was either maintained or raised to 10 600 ϩ mM after a baseline was established. When [K ]o was raised to 10 K 10 mM, there was a slight increase in outward current seen in 500 some but not all experiments, despite a decreased driving force, 400 http://ajprenal.physiology.org/ and the level was well maintained for 2 min, and there was a robust inhibition by TPNQ at the end of this period. In contrast, 300

ϩ pA in the continued presence of 1 mM [K ]o the outward current decreased progressively with only a small response to TPNQ 200 after 2 min. Figure 3B summarizes the results of this and ϩ TPNQ similar experiments. As in the previous protocol, 1 mM [K ]o 100 suppressed ISK. ϩ Although 1 mM [K ]o clearly inhibited ROMK channels in 0 the protocols of both Figs. 2 and 3, several aspects of the 0 20 40 60 80 100 120 140 results were puzzling. First, the baseline outward currents in s ϩ cells preincubated in 1 or 10 mM [K ]o were not very different by Henry Sackin on July 30, 2013 (compare initial currents in Figs. 2 and 3). Second, even though B Form whole-cell ϩ + the cells in Fig. 3 were preincubated in low [K ]o, maintaining in 1 mM [K ]o this concentration after the whole-cell clamp was formed led to ϩ + 1 mM or 10 mM [K ]o a decline in the current. Thus it appeared that reduced [K ]o 1.2 had a stronger effect on the channels under whole-cell condi- for 2 min tions than when the cells were intact. To test this further, we add TPNQ measured TPNQ-sensitive currents in paired cells of the same 1 tubule after preincubation for at least 5 min in either 10 or 1 ROMK activity ϩ mM [K ]o. The order was alternated to account for time- * dependent changes in channel activity. There was no discern- 0.8 ϩ ible effect of [K ]o under these conditions (Fig. 4). This 1K->10K suggests that conditions influenced by the formation of the whole-cell clamp predispose the channels to downregulation 0.6 1K->1K ϩ by low [K ]o. While the whole-cell conditions are unphysiological since cytoplasmic constituents are removed and replaced with those 0.4 of the pipette solution, we felt that examination of what factors normalized current ϩ are involved in the [K ]o -dependent downregulation of the 0.2 channels might give some insight into the physiological regulation of the channels. We examined several possible factors that might exert such an influence as shown in Fig. 5. 0 First, as mentioned in the Introduction, in oocytes inhibition initial final TPNQ ISK ϩ by [K ]o depends on the intracellular pH, and the effect of low Fig. 3. Effect of increasing [Kϩ] on ROMK currents in the rat CCD. ϩ o [K ]o appears as a shift in the pH dependence such that the A: whole-cell recording showing outward currents at a holding potential of 0 ϩ mV. Whole-cell clamps were formed in the presence of 1 mM [K ]o. Currents channels are closed with a more alkaline pKi. To evaluate ϩ ϩ whether this could account for the observations in Figs. 2 and declined with 1 mM [K ]o but stabilized in the presence of 10 mM [K ]o.At the end of the recording, TPNQ (100 nM) was added to the medium to measure 3, we increased the pH of the pipette solution, and presumably the ROMK-specific currents. B: summary of 5 similar pairs of experiments. also of the cytoplasm that is dialyzed by this solution under Data represent means Ϯ SE. *P Ͻ 0.05, significant difference. whole-cell conditions, to 8.0. In oocytes this fully opens the pH

AJP-Renal Physiol • doi:10.1152/ajprenal.00185.2013 • www.ajprenal.org LUMINAL Kϩ AND ROMK CHANNELS IN CCD F211

500 covery is greatly accelerated by addition of a reducing agent such as DTT (30, 46). We therefore examined the effect of ␮ 400 inclusion of DTT (500 M) in the pipette solution on the ϩ response to [K ]o. As seen in Fig. 5, DTT abrogated the ϩ response to 1 mM [K ]o applied during whole-cell recording. 300 This suggests that oxidative stress induced by dialysis of the ϩ cell with pipette solution combines with low [K ]o to close the 200 channels. To test this further, we incubated intact tubules with an oxidiz- ISK (pA/cell) ing agent, H2O2, added to the bath. This compound was previ- 100 ously shown to inhibit ROMK channels in cell-attached patches from the CCD (40). TPNQ-sensitive currents were then measured 0 immediately after formation of whole-cell conditions to assess the 10 K 1K response of the intact cell to the oxidant. As shown in Fig. 6, Downloaded from Fig. 4. TPNQ-sensitive outward currents (ISK) in paired principal cells after exposure of the cells to H2O2 at a concentration of 100 ␮M for Ͼ ϩ Ϯ ϩ preincubation for 5minin1or10mM[K ]o. Data represent means SE 10–20 min reduced I ina[K ] -dependent manner. With 10 ϩ SK o for 10–11 measurements. Incubation of intact cells in low [K ]o. did not ϩ ϩ mM [K ]o, the effect was minimal, while in 1 mM [K ]o. there diminish ROMK currents. ϩ was a consistent and dramatic inhibition. The higher [K ]o does not completely abolish the response to the oxidant; higher con- ϩ Ն gate of the channels even with 1 mM [K ]o (29). The response centrations ( 1 mM) of H2O2 strongly decreased ISK even at 10 ϩ ϩ mM [K ]o (data not shown). Nevertheless, these results conﬁrm to1mM[K ]o under these whole-cell clamp conditions was http://ajprenal.physiology.org/ somewhat reduced but was still evident (Fig. 5). We therefore the sensitivity of the channels to an oxidizing agent and demon- ϩ decided to pursue other possible mechanisms. strate that high [K ]o protects the channels against this inhibition. In excised inside-out patches ROMK channels run down We then examined the possibility that changes in the mem- rapidly. The loss of activity can be slowed by a cocktail of brane content of phosphatidylinositol bisphosphate (PIP2) may phosphatase inhibitors (FVPP solution) containing FϪ, vana- be involved in these responses. This phospholipid stabilizes the date, and pyrophosphate (20). To our surprise, this solution by activity of ROMK (15, 20), and oxidative stress has been itself appeared to reduce ROMK currents under whole-cell shown to activate its breakdown through activation of PLC (26, conditions. We do not understand this difference between the 38). Tubules were preincubated for 10 min or more with PLC whole-cell and excised-patch conﬁgurations. However, even inhibitor U73122 (33) at a concentration of 5 ␮M. This ϩ with these reduced currents an inhibitory effect of low [K ]o compound was previously shown to prevent the stimulation of

was documented (Fig. 5), suggesting that phosphatase activity TRPC3 channels by t-BHP, an effect thought to involve acti- by Henry Sackin on July 30, 2013 ϩ ␥ is not essential for the observed [K ]o dependence. vation of PLC and depletion of plasma-membrane PIP2 (14). ROMK channels in excised patches from oocytes are sensi- Whole-cell clamps were then established in the presence of 10 ϩ tive to redox conditions. In particular, when the channels are mM [K ]o and maintained for 2 min with either 10 or 1 mM ϩ closed by low pH on the cytoplasmic side of the patch, re- [K ]o. ISK was then assessed by addition of TPNQ. As shown in Fig. 5, PLC inhibition did not have a strong effect on the baseline ROMK current but abolished the inhibition observed 600 ϩ with low [K ]o. We then tested whether these effects could be reproduced in 10K 500 Xenopus oocytes expressing ROMK channels. This system is 1K

400 350

300 control 300 H O 2 2 250 * * ISK (pA/cell) 200 200

150 100

* ISK (pA/cell) 100 0 control pH 8 FVPP DTT U73122 50

ϩ * Fig. 5. [K ]o-dependent decreases in ROMK current. TPNQ-sensitive outward currents were measured after maintaining the whole-cell clamp for 2 min with 0 ϩ 10 K 1 K [K ]o ϭ 1 or 10 mM. Differences in current persisted with pH 8.0 in the ϩ pipette solution and with FVPP pipette solution. Inclusion of 0.5 mM DTT in Fig. 6. H2O2 decreases ROMK current in a [K ]o-dependent manner. TPNQ- the pipette solution or preincubation of tubules with U73122 (5 ␮M) abolished sensitive outward currents were measured in cells preincubated for Ͼ10 min ϩ the differences. Data represent means Ϯ SE for 17 pairs of cells (control), 13 with [K ]o ϭ 1 or 10 mM with or without 0.1 mM H2O2. Data represent pairs (pH 8), 9 pairs (FVPP), 18 pairs (DTT), or 11 pairs (U73122). *P Ͻ 0.05, means Ϯ SE for 4 experiments, with measurements in 2–3 cells for each signiﬁcant difference. condition. *P Ͻ 0.05, signiﬁcant difference.

AJP-Renal Physiol • doi:10.1152/ajprenal.00185.2013 • www.ajprenal.org F212 LUMINAL Kϩ AND ROMK CHANNELS IN CCD

Fig. 7. Effect of t-BHP on whole-cell currents in Kir1.1b (ROMK2)-expressing Xenopus ϩ oocytes with [K ]o ϭ1.0 mM, pHi ϳ 7.8. A: representative current traces, measured in response to voltages applied between Ϫ110 and ϩ50 mV. Dashed line denotes zero current. Upward deﬂections from the dashed line denote outward current. B: current-voltage re- lations recorded in the same oocyte at different ␮ times after application of 500 M tert-butyl Downloaded from hydroperoxide (t-BHP). http://ajprenal.physiology.org/

ϩ more stable than the principal cell under whole-cell voltage clamp [K ]o concentrations. Figure 7 shows current traces and I–V ϩ and enables the continuous measurement of ROMK-mediated relationships measured in the presence of 1 mM [K ]o before and currents since the background conductance is very low. We ﬁrst at various times after addition of t-BHP. There was a progressive examined the effects of H2O2 added to the bathing medium. In fall in outward currents to a level ϳ50% of baseline after 50 min. ϩ agreement with previous results (3), this produced no substantial Figure 8 illustrates a similar experiment with 0.1 mM [K ]o Here inhibition of the channels, even when used at higher concentra- the inhibition by t-BHP was faster and more complete. A sum- ϩ tions (1 mM) and for longer times (up to 50 min) than were used mary of the effects of t-BHP with the three different [K ]o is with the CCD. This could have been due to a low permeability of plotted in Fig. 9. These results suggest that ROMK channels are the oocyte plasma membrane to the reagent. We therefore tested affected by oxidizing agents in the CCD and in the oocyte by by Henry Sackin on July 30, 2013 t-BHP, a membrane-permeant analog of H2O2. At a concentration similar mechanisms. of 500 ␮M, this compound did not affect ROMK currents in 10 To examine the role of PLC in this system, oocytes incu- ϩ ϩ mM [K ]o. However, substantial inhibition was observed at lower bated in 1 mM [K ]o were treated with 500 ␮M t-BHP in the

Fig. 8. Effect of t-BHP on whole-cell currents in Kir1.1b (ROMK2)-expressing Xenopus ϩ oocytes with [K ]o ϭ0.1 mM, pHi ϳ 7.8. A: representative current traces, measured in response to voltages applied between Ϫ100 and ϩ40 mV. Dashed line denotes zero current. Upward deﬂections from the dashed line denote outward current. B: current-voltage re- lations recorded in the same oocyte at different times after application of 500 ␮M t-BHP.

AJP-Renal Physiol • doi:10.1152/ajprenal.00185.2013 • www.ajprenal.org LUMINAL Kϩ AND ROMK CHANNELS IN CCD F213

Fig. 9. Effect of t-BHP (500 ␮M) on outward conduc- ϩ tance in ROMK2-expressing oocytes with [K ]o ϭ 10, 1, and 0.1 mM. Outward conductance was normalized to that measured just before addition of t-BHP. ϩ Data are means Ϯ SE for n ϭ 5 oocytes at each [K ]o. Acetate buffers maintained pHi ϭ7.8, throughout. Downloaded from http://ajprenal.physiology.org/ presence and absence of U73122 (Fig. 10). The addition of 10 tively charged phospholipid stabilizes the open state ROMK ␮M U73122 nearly abolished the response to t-BHP. In con- (15, 20), as well as that of other inward rectifiers (8), by trast, a related compound U73343 that lacks activity against interacting with positively charged lysine and arginine residues PLC (33) did not interfere with the inhibition by t-BHP. This situated at the membrane/cytoplasm interface. is consistent with results with the CCD and suggests that Molecular mechanism. It is likely that oxidation, low-K, and reduction in membrane PIP2 content represents at least one PIP2 effects are all related. The simplest mechanism involves mechanism through which oxidative stress can modulate ϩ ϩ binding of K to sites at the outer mouth of the selectivity ROMK channels in a [K ]o-dependent manner. filter, stabilizing it in an open configuration. This interaction would antagonize any allosteric effects that favor a closed state DISCUSSION in which the filter collapses (e.g., Ref. 47). In this way external ϩ ϩ Kϩ could antagonize a variety of inhibitory effects that act on by Henry Sackin on July 30, 2013 Effects of [K ]o. External K has several effects on ROMK ϩ channels, all of them stimulatory. Increasing [K ]o shifts the the inside of the cell including cytoplasmic acidification, PIP2 pH dependence of the channels, keeping them open in more depletion, and oxidative stress. Consistent with this idea, sev- ϩ acid conditions (29, 31). Furthermore, high [K ]o prevents the eral Kir channels including Kir1.1 (ROMK) and 4.1 are selec- channels from entering a stable inactive state in the presence of tively activated by cations that interact with the pore either as low intracellular pH (27). In this study, we show that 10 mM permeant ions or blockers (7, 9, 29). In this respect, regulation ϩ ϩ [K ]o also protects the channels from closure in response to of Kir channels by external K is analogous to C-type inacti- oxidative stress. This appears to involve, at least in part, a vation of voltage-gated Kϩ channels, a process that is also reduction in the amount of PIP2 in the membrane. This nega- inhibited by extracellular permeant ions (18, 21).

Fig. 10. Effect of PLC inhibition (U73122) on the re- ϩ sponse to t-BHP with [K ]o ϭ 1 mM. Oocytes were treated with 500 ␮M t-BHP in the presence of 10 ␮M U73122, U73343, or without either drug. Outward conductance was normalized to that measured just before the addition of t-BHP. Data are means Ϯ SE for 4 oocytes under each condition. Acetate buffer maintained pHi ϭ 7.8 throughout.

AJP-Renal Physiol • doi:10.1152/ajprenal.00185.2013 • www.ajprenal.org F214 LUMINAL Kϩ AND ROMK CHANNELS IN CCD

ϩ Similar effects of H2O2 on ROMK channels measured in (34, 45). In contrast, the effects of low [K ]o described here cell-attached patches from rat CCD were previously reported would serve to inhibit the channels remaining in the luminal (40). In that study inhibition of channel activity was also membrane. Furthermore, a low-K diet enhanced the response purported to be indirect, rather than through oxidation of the of the CCD to inhibition by H2O2 in vitro (40). This chronic channel protein itself, since the effect was not observed in effect of K-depletion is distinct from the acute sensitization by ϩ excised patches and could be prevented by simultaneous treat- low ambient [K ]o reported here. Finally, a low-K diet predis- ment with antagonists of tyrosine kinase, P38, and ERK path- poses the channels to inhibition by ANG II in vitro (41). The ways. However, this phenomenon was observed with the chan- mechanism that we propose here through which ROMK may nels exposed to high (140 mM) external Kϩ and the mecha- be influenced by ANG II in vivo would be superimposed with nism may therefore be different from that reported here. this effect. The presence of multiple mechanisms for prevent- Physiological significance. The main goal of this investiga- ing Kϩ secretion when urinary K losses need to be minimized tion was to test whether external Kϩ could regulate ROMK underscores the importance to the organism of maintaining K channels in renal tubules and over a physiological concentra- balance. tion range. This range is actually quite large; during antidiure- sis, luminal Kϩ concentrations in the outer medullary collect- GRANTS Downloaded from ing duct exceed 40 mM (42). We were more interested, This work was supported by National Institute of Diabetes and Digestive however, in the lower end of the spectrum. Micropuncture and Kidney Diseases Grants R01-DK-27847 (to L. G. Palmer) and R01-DK- measurements showed that luminal Kϩ in the DCT is main- 46950 (to H. Sackin). Ͻ tained at 2 mM when dietary K intake is low but increases to DISCLOSURES 10–30 mM in the distal half of the DCT, with a normal K intake (23). Here the “DCT” was defined as the part of the No conflicts of interest, financial or otherwise, are declared by the author(s). http://ajprenal.physiology.org/ distal nephron accessible to micropuncture and may include AUTHOR CONTRIBUTIONS Kϩ-secreting segments such as the DCT2 and connecting tubule. We therefore considered the question of whether Author contributions: G.F., H.S., and L.G.P. conception and design of ϩ research; G.F., H.L., H.S., and L.G.P. performed experiments; G.F., H.S., and changes in [K ]o from 1 to 10 mM could directly influence L.G.P. interpreted results of experiments; G.F., H.S., and L.G.P. edited and channel activity. The main finding was that indeed regulation revised manuscript; G.F., H.L., H.S., and L.G.P. approved final version of could occur over this concentration range at least under con- manuscript; H.S. and L.G.P. analyzed data; H.S. and L.G.P. prepared figures; ditions of oxidative stress. When this occurs, channel activity L.G.P. drafted manuscript. ϩ ϩ would be shut down when luminal [K ] is low, minimizing K REFERENCES secretion even when the driving force exerted by the concentration gradient across the apical membrane is large. 1. Babilonia E, Li D, Wang Z, Sun P, Lin DH, Jin Y, Wang WH. Mitogen-activated protein kinases inhibit the ROMK (Kir 1.1)-like small The oxidative stress imposed by the whole-cell clamp con- conductance K channels in the cortical collecting duct. J Am Soc Nephrol by Henry Sackin on July 30, 2013 ditions may involve the removal of reducing agents from the 17: 2687–2696, 2006. cytoplasm as the constituents are exchanged with the solution 2. 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AJP-Renal Physiol • doi:10.1152/ajprenal.00185.2013 • www.ajprenal.org Inhibition of ROMK channels by low extracellular K+ and oxidative stress Gustavo Frindt, Hui Li, Henry Sackin and Lawrence G. Palmer Am J Physiol Renal Physiol 305:F208-F215, 2013. First published 15 May 2013; doi: 10.1152/ajprenal.00185.2013

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