ROMK is required for expression of the 70-pS K channel in the thick ascending limb

【摘要】  Apical potassium recycling is crucial for salt transport by the thick ascending limb (TAL). Loss-of-function mutations in the K channel, ROMK (Kir1.1; KCNJ1 ), cause Bartter syndrome, a genetically heterogeneous disorder characterized by severe reduction in salt absorption by the TAL, Na wasting, polyuria, and hypokalemic alkalosis. ROMK(-/-) null mice exhibit a Bartter phenotype and lack the small-conductance (30-pS) apical K channel (SK) in the TAL. However, a distinct 70-pS K channel can also significantly contribute to the apical conductance of TAL. We now examine the effect of ROMK deletion on the functional expression of the 70-pS K channel in the TAL. Functional expression of the 70-pS K channel was low [average channel acitivty ( NP o ) = 0.02] in ROMK(+/+) mice on a control K diet but increased to 0.27 by high-K intake for 2 wk. In contrast, the high-K diet decreased NP o of SK by 30%, from 2.04 to 1.44. In ROMK heterozygous (+/-) mice on a control K diet, SK activity was about one-half of that observed in ROMK(+/+) mice (0.95 vs. 2.04). The high-K diet also reduced SK activity in ROMK(+/-) mice by 40% (from 0.95 to 0.55) but increased NP o of the 70-pS K channel from 0 to 0.09 in ROMK(+/-) mice. This corresponds to 30% of channel activity ( NP o = 0.27) observed in ROMK(+/+) mice. Neither the 70-pS nor the 30-pS K channels were observed in TAL cells from ROMK(-/-) mice on either the normal or high-K diets. Thus functional expression of the 70-pS K channel is enhanced by increasing dietary K and requires expression of ROMK. It is likely that ROMK forms a critical subunit of the 70-pS K channel, accounting for the loss of apical K secretory channel activity in ROMK Bartter syndrome.

small-conductance potassium channel; intermediate-conductance potassium channel; Kir 1.1; Bartter syndrome

【关键词】  required expression ascending

K RECYCLING THROUGH APICAL K channels in the thick ascending limb (TAL) not only generates a lumen-positive voltage but also provides K for luminal Na-K-2Cl cotransport ( 3, 24 ). ROMK (Kir1.1) channels share many functional and regulatory properties with the 30-pS K channel (SK) in apical membranes of both TAL and principal cells ( 11, 12, 20 ). Consistent with a role of the ROMK channel in TAL function, mutations in the ROMK gene ( Kcnj1 ) cause type II Bartter syndrome characterized by Na wasting, polyuria, and hypokalemic metabolic alkalosis ( 9, 22 ). ROMK null mice exhibit a phenotype of Bartter syndrome, including both Na and K wasting and volume depletion ( 13, 17 ). We recently reported that SK channels were absent in TAL cells from ROMK null mice, establishing the role of ROMK in forming the native SK channel and the importance of apical K recycling in this nephron segment ( 17 ).

An unresolved issue in understanding the role of ROMK in Bartter syndrome and K recycling is the role of an intermediate-conductance (60-70 pS) K channel that contributes up to 70% to the apical conductance in the TAL ( 18, 24 ). Loss-of-function mutations of ROMK would not be expected to produce a severe Bartter's phenotype unless ROMK expression was required for function of the 70-pS K channel.

Although 70-pS K channels were not observed previously in the apical membrane of the TAL in ROMK null mice, no conclusion could be reached about the role of ROMK in forming the intermediate-conductance K channel because mice were kept on a control K diet in which the 70-pS K channel is very poorly expressed ( 17 ). However, it has been reported that raising dietary K increases the activity of the 70-pS K channel in the TAL ( 6 ). Thus we assessed both the effects of increasing dietary K intake and the role of the ROMK gene on the functional activities of both the SK and the 70-pS K channels in apical membranes of TAL cells from wild-type (+/+), heterozygous (+/-), and homozygous (-/-) ROMK null mice. Our results demonstrate that dietary K is an important regulator of both the intermediate-conductance and SK channels and that ROMK is required for functional expression of the 70-pS K channel in TAL cells.

METHODS

The generation of the ROMK null mutant mice was described previously ( 13, 17 ). We used ROMK(+/-) heterozygous breeding pairs to generate ROMK genotypes. Pups were genotyped 7 days after birth by PCR using DNA extracted from tail biopsies. All mice were 4-6 wk old and were maintained on standard (0.62%) or high-K (10%) mouse chow (Harlan Teklad, Madison, WI) for 14 days and tap water until the day of the experiment. All protocols were approved by Yale University's Animal Use and Care Committee.

Tubule preparation. The left kidney was removed after anesthesia/euthanasia by intraperitoneal injection of pentobarbital sodium (0.1 mg/g body wt) and cut into slices. Cortical TAL tubules were dissected in chilled bath solution, immobilized on a 5 x 5-mm cover glass coated with Cell-Tak (Biopolymers, Farmington, CT), and transferred to a patch chamber mounted on the stage of an inverted microscope (Olympus IMT-2). The tubule lumen was opened by a sharpened micropipette to expose the apical surface of the cells for patch clamping. All experiments were carried out at room temperature (22-24°C).

Patch clamping. In general, patch clamping was performed as described previously ( 18 ). Briefly, glass pipettes were pulled from borosilicate glass capillaries (Dagan, Minneapolis, MN) using a two-step Narishige PP83 puller (Narishige, Tokyo, Japan) and polished to give a pipette resistance from 6 to 8 M when filled with 140 mM KCl solution. Single-channel currents were amplified by an EPC-7 amplifier (List Electronics, Darmstadt, Germany) and low-pass filtered at 1 kHz by an eight-pole Bessel filter (902LPF; Frequency Devices, Haverhill, MA). Signals were digitized at a sampling rate 4 kHz (DigiData 1200; Axon Instruments, Foster City, CA) and stored for later analysis (Gateway2000, E-3100). Bath solution contained (in mM) 140 NaCl, 5 KCl, 1.8 MgCl, 1.8 CaCl 2, and 10 HEPES and adjusted pH to 7.4 with NaOH. Pipette solution contained (in mM) 140 KCl, 1.8 MgCl 2, and 10 HEPES with pH adjusted to 7.4 with KOH. Data for the open probability of the average channel activity ( NP o ) calculations were obtained from detached inside-out patches.

Electrophysiology data analysis. Data were analyzed using pCLAMP software (version 6.0.4; Axon Instruments). In patches with channels, channel activity was assessed as described previously ( 14 )

where t i is the fractional open time spent at each of the observed current levels. Channel expression in the three ROMK genotypes for the two diets is described as

where A is the number of patches with active channels and B is the total number of giga seal patches with or without channel activity, or as

We previously validated this method of determining channel expression in these mice ( 17 ). Channel conductance was estimated from linear regression analysis of single channel current-voltage curves. Voltage applied to the pipette was referenced to the bath potential (- V ).

Plasma electrolytes measurement. Littermate male 5-wk-old ROMK wild-type (+/+) and knockout (-/-) mice were fed normal or high-K diets for 14 days. Electrolyte concentrations were examined in plasma obtained by retro-orbital puncture that represents a mixed arterial-venous sample. Plasma was analyzed using a Corning Blood Gas analyzer.

Statistics. Data are shown as means ± SE. A significant difference was considered by t -test ( P < 0.05).

RESULTS

Effect of K diet on K channels in ROMK(+/+) mice. The results in Fig. 1 and Table 1 show that 70-pS K channel activity was low in the TAL from ROMK(+/+) mice on a normal K diet (0.62%). Only 1 of 44 giga-ohm seal patches exhibited 70-pS K channel activity. No 70-pS K channel activity was observed in either ROMK(+/-) or ROMK(-/-) mice. We examined the effect of a high-K intake (10%) on channel activity, since previous studies had shown this enhances the activity of both SK and 70-pS K channels in rat medullary TAL cells ( 6, 7 ). Increasing dietary K did not significantly elevate plasma K (4.49 ± 0.13 mM; 0.62% K diet vs. 4.80 ± 0.20 mM, 10% K diet; P 0.05, n = 10). The NP o on the high-K diet was 1.17 ± 0.14; a change in NP o with the high-K diet could not be evaluated, since only one patch exhibited the 70-pS K channel on the normal K diet. However, a high-K intake increased the probability of finding the 70-pS K channel, since 13 of 57 patches exhibited the intermediate-conductance K channel ( Fig. 1 and Table 1; the probability of finding channels increased from 2 to 23%) and NP o x ( A / B ) from 0.02 to 0.27. This suggests that 70-pS K channel activity is regulated by K intake.

Fig. 1. Effect of K diet on the probability of observing small-conductance K (SK, 30 pS) and intermediate-conductance K (70 pS) channels in patches from ROMK(+/+) and ROMK(+/-) mice.

Table 1. Effect of (10%; g/g) K diet on SK and 70-pS channel expression in apical patches from TAL

The high-K diet increased NP o of the SK channel from 3.58 ± 0.11 to 5.54 ± 0.35, consistent with our previous study ( 6 ). In contrast, the high-K intake decreased the probability of finding SK channels from 57% (25/44 patches) to 26% (15/57 patches; see Fig. 1 ) and decreased NP o x ( A / B ) from 2.04 (control K diet) to 1.44 (10% K diet) in ROMK(+/+) mice ( Table 1 ). Because the open probability ( P o ) of both SK ( P o = 0.88 ± 0.09) and the 70-pS K channel ( P o = 0.59 ± 0.2) was not altered by diet, it is apparent that the high-K diet lowered the number of SK channels but increased that of 70-pS K channels.

A patch exhibiting both SK and 70-pS K channels is shown in Fig. 2 A together with their respective current-voltage ( I-V ) curves ( Fig. 2 B ). The characteristics of the 70-pS K channels in ROMK(+/+) mice were identical to those previously observed in Sprague-Dawley rats and DC-1 mice ( 15, 27 ). The inward current of the 70-pS K channel displayed flickery kinetics with brief closures in the cell-attached configuration and showed two current levels, O 1 and O 2, representing at least two open 70-pS K channels in this patch. The slope conductance of the channels exhibiting the larger currents (as shown in Fig. 2, B and C ) was 70 ± 4 pS between 20 mV and -20 mV with two open times (0.5 ± 0.1, 14 ± 2 ms) and one closed time (3.6 ± 0.2 ms). The P o of the 70-pS channel was 0.59 ± 0.2 ( n = 7 patches). The I-V curve summarized in Fig. 2 B shows that SK channels had an inward slope conductance of 30.7 ± 0.5 pS between -40 and -80 mV (17.6 ± 1.2 pS between 0 and 20 mV), and it was not significantly different compared with SK channels in ROMK(+/+) on the normal K diet ( 17 ) and SK channels in TAL or principal cells of Sprague-Dawley rats ( 1, 4, 21 ).

Fig. 2. A : recording showing the activity of the 30-pS and the 70-pS K channel in a cell-attached patch from ROMK(+/+) mice on the 10% K diet. Inward current (downward deflection) of the 70-pS K channel is indicated by dotted lines (O 1 and O 2 ). Arrow indicates the opening of the SK channel (30 pS). C is the channel closed line. The holding potential is indicated on left. B : current ( I )-voltage ( V ) curve showing the relationship between voltage and channel current of SK and 70-pS channels. C : single-channel recording demonstrating the kinetics of the 70-pS K channel.

Effect of K diet on K channels in ROMK(-/-) mice. The ROMK(-/-) mice placed on the high-K diet for 14 days developed mild hyperkalemia (4.82 ± 0.15 for 0.62% K diet vs. 5.44 ± 0.16 for 10% K diet, P < 0.05, n = 8). Moreover, we confirmed the previous findings demonstrating that the SK channel is absent in principal cells in ROMK(-/-) on a control K diet ( 17 ). Importantly, neither SK nor 70-pS K channels were observed in the TAL of ROMK(-/-) mice receiving the 10% K diet ( Fig. 1 and Table 1 ). These findings confirm that ROMK encodes the SK channel and further establish a possible role for ROMK in forming the intermediate-conductance K channel.

Effect of K diet on K channels in ROMK(+/-) mice. We also examined the effect of the 10% K diet on the expression of the SK and 70-pS K channels in ROMK(+/-) mice in which only one of the ROMK alleles had been deleted. The high-K diet increased the NP o of the SK channel from 2.89 ± 0.23 to 3.92 ± 0.60, consistent with the observations in the ROMK(+/+) mice. However, the percent of patches with SK channels [( A / B ) x 100%; Table 1 and Fig. 1 ] was reduced by 57%, from 33% (control K diet) to 14% (10% K), consistent with an expected 50% reduction in protein expression in these ROMK heterozygous mice ( 17 ). Similarly, the high-K diet reduced the NP o x ( A / B ) of the SK channel ( Table 1 ) from 0.95 (control K diet) to 0.55 (high-K diet). If the SK and 70-pS K channels were formed from different genes and functioned independently, loss of one ROMK (SK) allele and a reduction in SK activity would not be expected to alter the activity of the 70-pS K channel. However, the probability of finding the 70-pS K channel [( A / B ) x 100%; Table 1 ] in mice fed the high-K diet was also decreased by 56%, from 23% (13 of 57 patches) in ROMK(+/+) mice to 10% (6 of 63 patches) in ROMK(+/-) mice. Moreover, NP o x ( A / B ) is similarly reduced from 0.27 in ROMK(+/+) mice to 0.09 in ROMK(+/-) mice. Thus loss of one ROMK allele reduces the activity of the 70-pS K channel by 50% in ROMK(+/-) mice on a high-K diet.

The decrease in 70-pS K channel activity is most likely the result of a reduction in channel density because the mean P o calculated from patches is similar between ROMK(+/+) and ROMK(+/-) mice ( P o = 0.59 ± 0.2 and 0.58 ± 0.4, respectively). Figure 3 is a typical recording showing the activity of a 70-pS K channel in the TAL from ROMK(+/+) and (+/-) mice on a high-K diet. Although the probability of finding the 70-pS K channels decreased by 50% in ROMK(+/-) mice compared with that in wild-type mice (see above), it is apparent that the NP o of the two patches from ROMK(+/+) and ROMK(+/-) mice were similar ( Table 1 ). Taken together, these results establish that ROMK is required for functional expression of the 70-pS K channel in the TAL.

Fig. 3. Recording demonstrating the activity of the 70-pS K channel in patches from ROMK(+/+) and ROMK(+/-) mice on a high-K diet. The holding potential was 0 mV, and the experiment was performed in a cell-attached patch.

DISCUSSION

It is well established that ROMK encodes the 30-pS K channel (SK) in the TAL and principal cells of the collecting duct. This conclusion is based on the observations that native SK channels and ROMK have similar functional and regulatory properties ( 20, 24 ). ROMK channels in the TAL play a key role in K recycling, which is essential for maintaining the activity of apical Na-K-Cl cotransport ( 5, 10 ). The importance of the coupling between ROMK and Na transport in the TAL has been further demonstrated by genetic studies in which loss-of-function mutations in ROMK (KCNJ1) were associated with type II Bartter syndrome characterized by massive loss of Na, Cl, and K in the urine ( 17 ).

Several K channels, the SK, 70-pS, and maxi-K channels, have been identified in the apical membrane of the TAL ( 26 ). However, it is generally accepted that the SK and the 70-pS K channels contribute most importantly to the apical K conductance and K recycling ( 24 ). Moreover, it has been suggested that the 70-pS K channel accounts for up to 70% of the total apical K conductance ( 18 ). This observation regarding the role of the 70-pS K channel in K recycling is difficult to reconcile with the finding that ROMK gene deletion affects severely NaCl reabsorption by the TAL ( 13, 17 ). One possible explanation is that ROMK is also involved in forming apical 70-pS K channels. This possibility was suggested previously by the absence of the 70-pS K channel in ROMK(-/-) mice. However, because 70-pS K channels could only rarely be identified in the ROMK(+/+) mouse, further studies in animals on a high-K diet were undertaken to examine the interaction between the SK and 70-pS K channels.

In the present study, 70-pS K channels were virtually absent in mice on the control K diet from all three ROMK genotypes, confirming our previous observations ( 17 ). The high-K diet increased expression of both the SK and 70-pS K channels in ROMK(+/+) and ROMK(+/-) mice, consistent with the study in rats ( 6 ). In contrast, the frequency of finding 70-pS K channels in ROMK(-/-) mice remained zero on the high-K diet. For independent expression of SK and 70-pS K channels, we would expect that the elimination of SK expression in ROMK(-/-) mice would have no effect on the high-K diet increasing 70-pS K channel expression. Thus functional expression of the 70-pS K channel requires ROMK.

Although the NP o of SK increased in ROMK(+/+) mice on the high-K diet, the frequency of finding this channel decreased by 50% on the high-K diet. Thus, in ROMK(+/+) mice, the high-K intake led to opposite changes in the functional expression of SK (decrease) and 70-pS K (increase) channels. In other words, a high-K intake changes the K channel type from predominantly SK to a mixture of SK and 70-pS K channels. Figure 4 shows a cell model of TAL cells and the effects of K intake on apical channel distribution. The simplest explanation for both the absence of 70-pS K channel activity in ROMK(-/-) mice and the 50% reduction of the frequency of finding SK channels in these mice is that ROMK is a subunit of the intermediate-conductance K channel. Because high dietary K intake has little effect on ROMK gene expression or channel protein content ( 19, 23 ), it is reasonable to suggest that incorporation of ROMK subunits into 70-pS K channels could reduce the frequency of finding functional SK channels while increasing that of intermediate-conductance K channels. This hypothesis would require the presence of a unique protein that interacts with ROMK to form the 70-pS K channel and that its expression is regulated by dietary K ( Fig. 4, bottom ). The mechanism by which ROMK would influence functional expression of the 70-pS K channel subunit could involve trafficking, gating, or some other undefined process. Identification of the specific mechanism will require cloning of the 70-pS K channel subunit. It should be noted that we cannot completely exclude potential influences of other factors in modulating SK and 70-pS K channel expression, but these factors would have to account for the 50% reduction in the frequency of finding ROMK and 70-pS K channel in ROMK(+/+) and ROMK(+/-) mice on either normal or high-K diets. However, the effects of differences in membrane potential, cell K activity, and basolateral-apical membrane cross talk in accounting for any observed differences in NP o values for SK or 70-pS K channel are unlikely since we obtained these data from excised patches.

Fig. 4. Top : cell model of thick ascending limb (TAL) illustrating the effect of changes in K diet on apical K channel distribution. Bottom : possible role of ROMK in forming the 70-pS K channel. It is proposed that dietary K intake increases the number of functional 70-pS K channels in TAL and renders the channel sensitive to 20-hydroxyeicosatetraenic (20-HETE), nitric oxide (NO), protein tyrosine kinase (PTK), and protein tyrosine phosphatase (PTP). PKA, protein kinase A; NKCC, Na-K-2Cl co-transporter.

The contribution of ROMK to the 70-pS K channel could account for its regulation by protein kinase A, ATP, arachidonic acid, and pH ( 24 ). Moreover, the channel component that differs from ROMK could confer other properties to the 70-pS K channel. In addition to the larger single-channel conductance, several factors may be involved in the specific activation of the 70-pS K channel by a high dietary K intake. 20-Hydroxyeicosatetraenic acid (20-HETE) is a potent inhibitor of the 70-pS K channel, and 20-HETE production has been shown to be lower in TAL cells from rats placed on a high-K diet ( 8 ). Furthermore, inhibition of 20-HETE production by cytochrome P -450 increases 70-pS K channel activity ( 6 ). Moreover, nitric oxide is a stimulator of the 70-pS K channel, and a high-K diet increases expression of inducible nitric oxide synthase in the renal medulla of rats ( 8, 16 ). Finally, channel protein phosphorylation and dephosphorylation by protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) also could contribute to regulation of the 70-pS K channel in rat K dietary intake. Expression of c-Src, a nonreceptor type of PTK, is higher in the renal medulla from rats on a K-deficient diet than from rats on a high-K diet ( 25 ). Inhibition of PTK enhances the activity of 70-pS K channels in rats on a K-deficient diet, whereas inhibition of PTP decreases K channel activity in the TAL from rats on a high (10%)-K diet ( 7 ). PTK/PTP regulation of apical K channels in the TAL is specific for the 70-pS K channel. Although Wang ( 28 ) has established that dietary K regulates SK channel density in principal cells by a PTK/PTP-based modulation of endocytosis of ROMK1, this ROMK isoform is not expressed in the TAL ( 2 ). Inducing PTP enhances only the 70-pS K channel but not SK channel activity, as expected from the absence of ROMK1 expression in the TAL ( 7 ). Because these factors are all regulated by dietary K intake, it is possible that the effect of K intake on apical K conductance in the TAL is achieved by a recombination of ROMK and 70-pS K channel protein subunits ( Fig. 4 ). In conclusion, the present studies provide strong evidence that ROMK is a component of the 70-pS K channel in the TAL.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54999 (S. C. Hebert), DK-54998 (G. Giebisch), and DK-54983 (W. Wang).

ACKNOWLEDGMENTS

We thank Dr. DeRen Shao for technical assistance with the collection of mouse blood samples.

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作者单位：1 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026; and 2 Department of Pharmacology, New York Medical College, Valhalla, New York 10595