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The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).

The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).

Ubiquitination influences the expression of the epithelial Na+ channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and Fisher rat thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaCs were strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see whether location of the protein in or near the apical membrane rather than cleavage per se influences ubiquitination, we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when coexpressed with α- and βENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed in situ surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total and ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na+ could stimulate ubiquitination. Administration of amiloride to block Na+ entry through the channels did not affect ubiquitination of γENaC in either FRT cells or the rat kidney. However, presumed large increases in cellular Na+ produced by monensin in FRT cells or acute Na+ repletion in rats increased ubiquitination and decreased overall ENaC expression. NEW & NOTEWORTHY: We have explored the mechanisms underlying the ubiquitination of the γ subunit of epithelial Na+ channel (ENaC), a process believed to control channel internalization and degradation. We previously reported that the mature, cleaved form of the subunit is selectively ubiquitinated. Here we show that this specificity arises not from the cleavage state of the protein but from its location in the cell. We also show that under some conditions, increased intracellular Na+ can stimulate ENaC ubiquitination. [ABSTRACT FROM AUTHOR]

The epithelial Na+ channel (ENaC) is essential for Na+ homeostasis, and dysregulation of this channel underlies many forms of hypertension. Recent studies suggest that mTOR regulates phosphorylation and activation of serum/glucocorticoid regulated kinase 1 (SGK1), which is known to inhibit ENaC internalization and degradation; however, it is not clear whether mTOR contributes to the regulation of renal tubule ion transport. Here, we evaluated the effect of selective mTOR inhibitors on kidney tubule Na+ and K+ transport in WT and Sgk1-/- mice, as well as in isolated collecting tubules. We found that 2 structurally distinct competitive inhibitors (PP242 and AZD8055), both of which prevent all mTOR-dependent phosphorylation, including that of SGK1, caused substantial natriuresis, but not kaliuresis, in WT mice, which indicates that mTOR preferentially influences ENaC function. PP242 also substantially inhibited Na+ currents in isolated perfused cortical collecting tubules. Accordingly, patch clamp studies on cortical tubule apical membranes revealed that mTOR inhibition markedly reduces ENaC activity, but does not alter activity of K+ inwardly rectifying channels (ROMK channels). Together, these results demonstrate that mTOR regulates kidney tubule ion handling and suggest that mTOR regulates Na+ homeostasis through SGK1-dependent modulation of ENaC activity.

The impact of chronic dietary K+ loading on proximal tubule (PT) function was measured using free-flow micropuncture along with measurements of overall kidney function, including urine volume, glomerular filtration rate, and absolute and fractional Na+ and K+ excretion in the rat. Feeding animals a diet with 5% KCl [high K+ (HK)] for 7 days reduced glomerular filtration rate by 29%, increased urine volume by 77%, and increased absolute K+ excretion by 202% compared with rats on a 1% KCl [control K+ (CK)] diet. HK did not change absolute Na+ excretion but significantly increased fraction excretion of Na+ (1.40% vs. 0.64%), indicating that fractional Na+ absorption is reduced by HK. PT reabsorption was assessed using free-flow micropuncture in anesthetized animals. At 80% of the accessible length of the PT, measurements of inulin concentration indicated volume reabsorption of 73% and 54% in CK and HK, respectively. At the same site, fractional PT Na+ reabsorption was 66% in CK animals and 37% in HK animals. Fractional PT K+ reabsorption was 66% in CK and 37% in HK. To assess the role of Na+/H+ exchanger isoform 3 (NHE3) in mediating these changes, we measured NHE3 protein expression in total kidney microsomes as well as surface membranes using Western blots. We found no significant changes in protein in either cell fraction. Expression of the Ser552 phosphorylated form of NHE3 was also similar in CK and HK animals. Reduction in PT transport may facilitate K+ excretion and help balance Na+ excretion by shifting Na+ reabsorption from K+-reabsorbing to K+-secreting nephron segments. NEW & NOTEWORTHY In rats fed a diet rich in K+, proximal tubules reabsorbed less fluid, Na+, and K+ compared with those in animals on a control diet. Glomerular filtration rates also decreased, probably due to glomerulotubular feedback. These reductions may help to maintain balance of the two ions simultaneously by shifting Na+ reabsorption to K+-secreting nephron segments. [ABSTRACT FROM AUTHOR]

Changes in apical surface expression of ion channels and transporters in the superficial rat renal cortex were assessed using biotinylation and immunoblotting during alterations in dietary K intake. A high-K diet increased, and a low-K diet decreased, both the overall and surface abundance of the [beta]- and [gamma]-subunits of the epithelial Na channel (ENaC). In the case of [gamma]-ENaC, the effect was specific for the 65-kDa cleaved form of the protein. The overall amount of [alpha]-ENAC was also increased with increasing K intake. The total expression of the secretory [K.sup.+] channels (ROMK) increased with a high-K diet and decreased with a low-K diet. The surface expression of ROMK increased with high K intake but was not significantly altered by a low-K diet. In contrast, the amounts of total and surface protein representing the thiazide-sensitive NaCl cotransporter (NCC) decreased with increasing K intake. We conclude that modulation of [K.sup.+] secretion in response to changes in dietary K intake involves changes in apical [K.sup.+] permeability through regulation of [K.sup.+] channels and in driving force subsequent to alterations in both Na delivery to the distal nephron and [Na.sup.+] uptake across the apical membrane of the [K.sup.+] secretory cells. K loading; K depletion; ENaC; ROMK; NCC doi: 10.1152/ajprenal.00323.2010.

The abundance of Na transport proteins in the luminal membrane of the rat kidney was assessed using in situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, the amounts of epithelial Na channel (ENaC) [beta]-subunit ([beta]-ENaC) and [gamma]-subunit ([gamma]-ENaC) and Na-Cl cotransporter (NCC) protein in the surface fraction increased relative to controls by 1.9-, 3.5-, and 1.5-fold, respectively. The amounts of the luminal Na/H exchanger (NHE3) and the luminal Na-K-2Cl cotransporter (NKCC2) did not change significantly. The increases in ENaC subunits were mimicked by administration of aldosterone for 1 wk, but the increase in NCC was not. When the animals were fed a high-Na (5% NaCl) diet for 1 wk, the surface expression of [beta]-ENaC increased by 50%, whereas that of the other membrane proteins did not change, relative to controls. The biochemical parameter most strongly affected by dietary Na was the abundance of the 65-kDa cleaved form of [gamma]-ENaC at the surface. This increased by 8.5-fold with Na depletion and decreased by 40% with Na loading. The overall 14-fold change reflected regulation of the total abundance of the subunit as well as the fraction of the subunit protein in the cleaved form. We conclude that cleavage of [gamma]-ENaC and its expression at the apical surface play a major role in the regulation of renal Na reabsorption. epithelial sodium channel; sodium-hydrogen exchanger isoform 3; sodium-potassium-chloride cotransporter; sodium-chloride cotransporter; sodium-deficient diet; sodium-rich diet doi: 10.1152/ajprenal.00401.2009.

Renal [Na.sup.+] and [K.sup.+] excretion was measured in rats with varying dietary [K.sup.+] intake. The requirement for channel-mediated distal nephron [Na.sup.+] reabsorption was assessed by infusing the animals with the [K.sup.+]-sparing diuretic amiloride via osmotic minipumps. At infusion rates of 2 nmol/min, the concentration of amiloride in the urine was 38 [micro]M, corresponding to concentrations of 9-23 [micro]M in the distal tubular fluid, sufficient to block >98% of [Na.sup.+] transport through apical [Na.sup.+] channels (ENaC). With a control [K.sup.+] intake (0.6% KC1), amiloride reduced [K.sup.+] excretion rates ([U.sub.k]V) from 0.85 [+ or -] 0.15 to 0.05 [+ or -] 0.01 [micro]mol/min during the first 2 h of infusion, suggesting that distal nephron [K.sup.+] secretion was completely dependent on the activity of [Na.sup.+] channels. When [K.sup.+] intake was increased by feeding overnight with a diet containing 10% KC1, amiloride reduced [U.sub.K]V from 7.5 [+ or -] 0.7 to 1.3 [+ or -] 0.1 [micro]mol/min despite an increased plasma [K.sup.+] of 9 mM, again suggesting a major but not exclusive role for the [Na.sup.+] channel-dependent pathway of [K.sup.+] secretion. The maximal measured rates of amiloride-sensitive [K.sup.+] excretion correspond well with estimates based on apical [K.sup.+] channel activity in distal nephron segments. However, when the animals were adapted to the high-[K.sup.+] diet for 7-9 days, the diuretic decreased [U.sub.K]V less, from 6.1 [+ or -] 0.6 to 3.0 [+ or -] 0.8 [micro]mol/min, indicating an increasing fraction of [K.sup.+] excretion that was independent of [Na.sup.+] channels. This indicates the upregulation of a [Na.sup.+] channel-independent mechanism for secreting [K.sup.+]. amiloride; [K.sup.+] adaptation; [K.sup.+] channels

The activity of ROMK channels in rat kidney tubule cells was assessed as tertiapin-Q (TPNQ)-sensitive current under whole cell clamp conditions. With an external [K.sup.+] concentration of 5 mM and an internal [K.sup.+] concentration of 140 mM and the membrane potential clamped to 0 mV, TPNQ blocked outward currents in principal cells of the cortical collecting duct (CCD) outer medullary collecting duct and connecting tubule (CNT). The apparent [K.sub.i] was 5.0 nM, consistent with its interaction with ROMK. The TPNQ-sensitive current reversed at voltages close to the equilibrium potential for [K.sup.+]. The currents were reduced when the pipette solution contained ATP. In the CCD, the average TPNQ-sensitive outward current ([I.sub.SK]) was 476 [+ or -] 48 pA/cell in control animals on a 1% KCl diet. [I.sub.SK] increased to 1,255 [+ or -] 140 pA when animals were maintained on a high-K (10% KCl) diet for 7 days and decreased to 314 [+ or -] 46 pA after 7 days on a low-K (0.1% KCl) diet. In the CNT, [I.sub.SK] was 360 -[+ or -] 30 pA on control, 1,160 [+ or -] 110 on high-K, and 166 [+ or -] 16 pA on low-K diets. The results indicate that ROMK channel activity is highly regulated by dietary K in both the CCD and the CNT. renal K channels; tertiapin-Q; K adaptation; K depletion

Inward-rectifier potassium (Kir) channels differ from the canonical [K.sup.+] channel structure in that they possess a long extended pore (~85 [Angstrom]) for ion conduction that reaches deeply into the cytoplasm. This unique structural feature is presumably involved in regulating functional properties specific to Kir channels, such as conductance, rectification block, and ligand-dependent gating. To elucidate the underpinnings of these functional roles, we examine the electrostatics of an ion along this extended pore. Homology models are constructed based on the open-state model of KirBac1.1 for four mammalian Kir channels: Kir1.1/ROMK, Kir2.1/IRK, Kir3.1/GIRK, and Kir6.2/KATP. By solving the Poisson-Boltzmann equation, the electrostatic free energy of a [K.sup.+] ion is determined along each pore, revealing that mammalian Kir channels provide a favorable environment for cations and suggesting the existence of high-density regions in the cytoplasmic domain and cavity. The contribution from the reaction field (the self-energy arising from the dielectric polarization induced by the ion's charge in the complex geometry of the pore) is unfavorable inside the long pore. However, this is well compensated by the electrostatic interaction with the static field arising from the protein charges and shielded by the dielectric surrounding. Decomposition of the static field provides a list of residues that display remarkable correspondence with existing mutagenesis data identifying amino acids that affect conduction and rectification. Many of these residues demonstrate interactions with the ion over long distances, up to 40 [Angstrom], suggesting that mutations potentially affect ion or blocker energetics over the entire pore. These results provide a foundation for understanding ion interactions in Kit channels and extend to the study of ion permeation, block, and gating in long, cation-specific pores.

Fejes-Toth G, Frindt G, Naray-Fejes-Toth A, Palmer LG. Epithelial [Na.sup.+] channel activation and processing in mice lacking SGK1. Am J Physiol Renal PhysioI 294: FI298-F1305, 2008. First published April 2, 2008; doi: 10.1152/ajprenal.00579.2007.--Amiloride-sensitive [Na.sup.+] channel activity was examined in the cortical collecting ducts of a mouse line ([SGK1.sup.-/-]) deficient in the serum-and glucocorticoid-dependent protein kinase SGK1. This activity was correlated with changes in renal Na handling and in the maturation of epithelial [Na.sup.+] channel (ENaC) protein. Neither [SGK1.sup.-/-] mice nor paired [SGK1.sup.+/+] animals expressed detectable channel activity, measured as amiloride-sensitive whole-cell current ([I.sub.Na]), under control conditions with standard chow. Administration of aldosterone (0.5 [micro]g/h via osmotic minipump for 7 days) increased [I.sub.Na] to a similar extent in [SGK1.sup.+/+] (378 [+ or -] 61 pA/cell at -100 mV) and in [SGK1.sup.-/-] (350 [+ or -] 57 pA/cell) animals. However, the maturation of ENaC, assessed as the ratio of cleaved to full-length forms of [gamma]-ENaC, was more pronounced in [SGK.sup.+/+] mice. The [SGK1.sup.-/-] animals exhibited a salt-wasting phenotype when kept on a low-Na diet for up to 2 days, losing significantly more Na in the urine than wild-type mice. Under these conditions, [1.sub.Na] was enhanced more in [SGK1.sup.-/-] (94 [+ or -] 14 pA/cell) than in [SGK.sup.+/+] (23 [+ or -] 5 pA/cell) genotypes. Despite the larger currents, the ratio of cleaved to full-length [gamma]-ENaC was lower in the knockout animals. The mice also expressed a smaller amount of [Na.sup.+]-[Cl.sup.-] cotransporter protein under Na-depleted conditions. These results indicated that SGK1 is essential for optimal processing of ENaC but is not required for activation of the channel by aldosterone. epithelial sodium channel; proteolysis; sodium reabsorption; cortical collecting duct

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface [beta] and [gamma]ENaC subunits were assayed by immunoblot, Surface [alpha]ENaC was assessed by membrane fractionation. Most of the [gamma]ENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.

The epithelial [Na.sup.+] channel (ENaC) is a mulfimeric membrane protein consisting of three subunits, [alpha], [beta], and [gamma]. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2[alpha]:1 [beta]:1[gamma] or a higher-ordered stoichiometry of 3[alpha]:3[beta]:3[gamma]. Therefore, while it is clear that all three ENaC subunits are required for full channel activity, the number of the subunits required remains controversial. We used a new approach, based on single-channel measurements in Xenopus oocytes to address this issue. Individual mutations that alter single-channel conductance were made in pore-lining residues of ENaC [alpha], [beta], or [gamma] subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of [alpha], [beta], and [gamma] showed a well-defined current transition amplitude with a single Gaussian distribution. When cRNAs for all three wild-type subunits were mixed with an equimolar amount of a mutant a-subunit (either $589D or S592T), amplitudes corresponding to pure wild-type or mutant conductances could be observed in the same patch, along with a third intermediate amplitude most likely arising from channels with at least one wild-type and at least 1 mutant [alpha]-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant [beta]G529A or [gamma]G534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2[alpha], 1[beta], and 1[gamma] come together around central pore.

The expression and activity of epithelial [Na.sup.+] channels (ENaC) in the medullary collecting duct of the rat kidney were examined using a combination of whole cell patch-clamp measurements of amiloride-sensitive currents ([I.sub.Na]) in split-open tubules and Western blot analysis of [alpha]-, [beta]-, and [gamma]-ENaC proteins. In the outer medullary collecting duct, amiloride-sensitive currents were undetectable in principal cells from control animals but were robust when rats were treated with aldosterone ([I.sub.Na] = 960 [+ or -] 160 pA/cell) or fed a low-Na diet ([I.sub.Na] = 440 [+ or -] 120 pA]cell). In both cases, the currents were similar to those measured in principal cells ofthe cortical collecting duct from the same animals. In the inner medullary collecting duct, currents were much lower, averaging 120 [+ or -] 20 pA/cell in aldosterone-treated rats. Immunoblots showed that all three ENaC subunits were expressed in the cortex, outer medulla, and inner medulla of the rat kidney. When rats were fed a low-Na diet for 1 wk, similar changes in [alpha]- and [gamma]-ENaC occurred in all three regions of the kidney; the amounts of full-length as well as putative cleaved [alpha]-ENaC protein increased, and the fraction of [gamma]-ENaC protein in the cleaved state increased at the expense of the full-length protein. The appearance of a presumably fully glycosylatedform of [beta]-ENaC in Na-depleted animals was observed mainly in the outer and inner medulla. These findings suggest that the capability of hormone-regulated, channel-mediated Na reabsorption by the nephron extends at least into the outer medullary collecting duct. ENaC; aldosterone; outer medulla; inner medulla

High-conductance (B K or maxi) [K.sup.+] channels were observed in cell-attached patches of the apical membrane of the isolated split-open rat connecting tubule (CNT). These channels were quite rare in cells identified visually as principal cells (PCs; 5/162 patches) but common in intercalated cells (ICs; 24/26 patches). The BK-expressing intercalated cells in the CNT and cortical collecting duct (CCD) were characterized by a low membrane potential (-36 mV) under short-circuit conditions, measured from the reversal potential of the channel currents with similar [K.sup.+] concentrations on both sides of the membrane. Under whole-cell clamp conditions with low intracellular [Ca.sup.2+], ICs had a very low [K.sup.+] conductance. When cell [Ca.sup.2+] was increased to 200 nM, a voltage-dependent, tetraethylammonium (TEA)-sensitive outward conductance was activated with a limiting value of 90 and 140 nS/cell in the CNT and CCD, respectively. Feeding animals a high-K diet for 1 wk did not increase these currents. TEA-sensitive currents were much smaller in PCs and usually below detection limits. To examine the possibility that the ICs participate in transepithelial [K.sup.+] secretion, we measured Na/K pump activity as a ouabain-sensitive current. Although these currents were easily observed in PCs, averaging 79 [+ or -] 14 and 250 [+ or -] 50 pA/cell in the CCD and CNT, respectively, they were below the level of detection in the ICs. We conclude that ICs have BK channel densities that are sufficient to support renal secretion of [K.sup.+] if cell [Ca.sup.2+] is elevated. However. a pathway for [K.sup.+] entry into these cells has not been identified. maxi-K channels; principal cells; intercalated cells; [K.sup.+] secretion; cortical collecting duct; connecting tubule

[Cl.sup.-] currents were observed under whole cell clamp conditions in cells of the rat cortical collecting duct (CCD), connecting tubule (CNT), and thick ascending limb of Henle's loop (TALH). These currents were much larger in intercalated cells compared with principal cells of the CCD and were also larger in the TALH and in the CNT compared with the CCD. The conductance had no strong voltage dependence, and steady-state currents were similar in inward and outward directions with similar [Cl.sup.-] concentrations on both sides of the membrane. Current transients were observed, particularly at low [Cl.sup.-] concentrations, which could be explained by solute depletion and concentration in fluid layers next to the membrane. The currents had a remarkable selectivity among anions. Among halides, [Br.sup.-] and [F.sup.-] conductances were only 15% of that of [Cl.sup.-], and [I.sup.-] conductance was immeasurably small. [SCN.sup.-] and [OCN.sup.-] conductances were ~50%, and aspartate, glutamate, and methanesulfonate conductance was ~5% that of [Cl.sup.-]. No conductance could be measured for any other anion tested, including N[O.sub.3.sup.-], HC[O.sub.3.sup.-], formate, acetate, or isethionate; N[O.sub.3.sup.-] and [I.sup.-] appeared to block the channels weakly. Conductances were diminished by lowering the extracellular pH to 6.4. The properties of the conductance fit best with those of the cloned renal anion channel ClC-K2 and likely reflect the basolateral [Cl.sup.-] conductances of the cells of these nephron segments. ClC-K2; anion selectivity; basolateral membrane; intercalated cells

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7-9 days, the abundance of two forms of the [alpha]-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the [beta]-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the [gamma]-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in [alpha]- and [gamma]-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in [alpha]- and [gamma]- but not [beta]-subunits when animals were treated with exogenous aldosterone, and in [beta]- and [gamma]- but not [alpha]-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney. Na channels; Na depletion; Na repletion; aldosterone; potassium intake; proteolytic cleavage doi:10.1152/ajprenal.00422.2005

Outward and inward currents through single small-conductance [K.sup.+] (SK) channels were measured in cell-attached patches of the apical membrane of principal cells of the rat cortical collecting duct (CCD). Currents showed mild inward rectification with high [[K.sup.+]] in the pipette ([K.sup.+.sub.p]), which decreased as [K.sup.+.sub.p] was lowered. Inward conductances had a hyperbolic dependence on [K.sup.+.sub.p] with half-maximal conductance at ~20 mM. Outward conductances, measured near the reversal potential, also increased with [K.sup.+.sub.p] from 15 pS ([K.sup.+.sub.p] = 0) to 50 pS ([K.sup.+.sub.p] = 134 mM). SK channel density was measured as the number of conducting channels per patch in cell-attached patches. As reported previously, channel density increased when animals were on a high-K diet for 7 days. Addition of 8-cpt-cAMP to the bath at least 5 rain before making a seal increased SK channel density to an even greater extent, although this increase was not additive with the effect of a high-K diet. In contrast, increases in Na channel activity, assessed as the whole cell amiloride-sensitive current, due to K loading and 8-cpt-cAMP treatment were additive. Single-channel conductances and channel densities were used as inputs to a simple mathematical model of the CCD to predict rates of transepithelial [Na.sup.a] and [K.sup.+] transport as a function of apical [Na.sup.a] permeability and [K.sup.+] conductance, basolateral pump rates and [K.sup.+] conductance, and the paracellular conductance. With measured values for these parameters, the model predicted transport rates that were in good agreement with values measured in isolated, perfused tubules. The number and properties of SK channels account for [K.sup.+] transport by the CCD under all physiological conditions tested. ROMK; epithelial sodium channels; high-potassium diet; aldosterone; cAMP; epithelial transport model

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at [V.sub.m] = -100 mV from 34 to 21 pS. The slope conductance measured between -60 and -140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by [Ba.sup.2+], suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for [T1.sup.+]:[K.sup.+], accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for [K.sup.+] transport. KEY WORDS: ROMK2 * IRK1 * GIRK1 * cytoplasmic pore * single-channel conductance

Apical potassium channels in the rat connecting tubule. Am J Physiol Renal Physiol 287: F1030-F1037, 2004. First published--; doi: 10.1152/ajprenal.00169.2004.--Apical membrane K channels in the rat connecting tubule (CNT) were studied using the patch-clamp technique. Tubules were isolated from the cortical labyrinth of the kidney and split open to provide access to the apical membrane. Cell-attached patches were formed on presumed principal and/or connecting tubule cells. The major channel type observed had a single-channel conductance of 52 pS, high open probability and kinetics that were only weakly dependent on voltage. These correspond closely to the 'SK'-type channels in the cortical collecting duct, identified with the ROMK (Kir1.1) gene product. A second channel type, which was less frequently observed, mediated larger currents and was strongly activated by depolarization of the apical membrane voltage. These were identified as BK or maxi-K channels. The density of active SK channels revealed a high degree of clustering. Although heterogeneity of tubules or of cell types within a tubule could not be excluded, the major factor underlying the distribution appeared to be the presence of channel clusters on the membrane of individual cells. The overall density of channels was higher than that previously found in the cortical collecting tubule (CCT). In contrast to results in the CCT, we did not detect an increase in the overall density of SK channels in the apical membrane after feeding the animals a high-K diet. However, the activity of amiloride-sensitive Na channels was undetectable under control conditions but was increased after both 1 day (90 [+ or -] 24 pA/cell) or 7 days (385 [+ or -] 82 pA/cell) of K loading. Thus one important factor leading to an increased K secretion in the CNT in response to increased dietary K is an increased apical Na conductance, leading to depolarization of the apical membrane voltage and an increased driving force for K movement out into the tubular lumen. ++ ROMK; BK; potassium adaptation; ENaC; sodium channels

ROMK channels are regulated by internal pH ([pH.sub.i]) and extracellular [K.sup.+] ([K.sup.+.sub.o]). The mechanisms underlying this regulation were studied in these channels alter expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular [H.sup.+], increasing the apparent pKa for inhibition by ~0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in [K.sup.+.sub.o], consistent with coupling between the responses to [pH.sub.i] and [K.sup.+.sub.o]. The ion selectivity sequence of the activation of the channel by cations was [K.sup.+] [congruent to] [Rb.sup.+] > N[H.sub.4.sup.+] >> [Na.sup.+], similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of [K.sup.+] from the cytoplasm and increasing sensitivity of the selectivity filter to removal of [K.sup.+.sub.o]. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With [K.sup.+.sub.o] between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular [K.sup.+] to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular [Ba.sup.2+] block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by [H.sup.+]. We conclude that decreasing [pH.sub.I] increases the sensitivity of ROMK2 channels to [K.sup.+.sub.o] by altering the properties of the selectivity filter. KEY WORDS: ROMK2 * IRK1 * renal K secretion * Xenopus oocytes * transmembrane helix

Frindt, Gustavo, and Lawrence G. Palmer. Na channels in the rat connecting tubule. Am J Physiol Renal Physiol 286: F669-674, 2004. First published December 16, 2003; 10.1152/ajprenal.00381.2003.--Epithelial Na channels were investigated using patch-clamp techniques in connecting tubule (CNT) segments isolated from rat kidney. Cell-attached patches with [Li.sup.+] in the patch pipette contained channels with conductances for inward currents of 13-16 pS and slow opening and closing kinetics, similar to properties of Na channels in the cortical collecting tubule (CCT). Macroscopic amiloride-sensitive currents ([I.sub.Na]) were also observed under whole cell clamp conditions. These currents were undetectable in cells from control rats but were large when the animals were infused with aldosterone (1,380 [+ or -] 340 pA/cell at a holding potential of--100 mV) or fed a high-K diet (670 [+ or-] 260 pA/cell) for 1 wk. Under both of these conditions, currents in cells of the CNT were two--to fourfold larger than currents in cells of the CCT of the same animals. In aldosterone-treated animals, currents in cells of the initial collecting tubule (iCT) were intermediate, such that the relative magnitude of [I.sub.Na] was as follows: CNT > iCT > CCT. Quantitative analysis of the results suggests that the maximal capacity of the aggregate population of CNTs to reabsorb Na could be as high as 18 [micro]mol/min, or ~10% of the filtered load of Na. This capacity is ~10 times higher than that of the CCT. epithelial sodium channel; amiloride; aldosterone; dietary potassium

Currents through epithelial Na channels (ENaCs) were measured in the cortical collecting tubule (CCT) of mice expressing truncated [beta]-subunits of ENaC, reproducing one of the mutations found in human patients with Liddle's syndrome. Tubules were isolated from mice homozygous for the Liddle mutation (L/L) and from wild-type (WT) littermates. Amilo-ride-sensitive currents ([I.sub.Na]) from single cells were recorded under whole cell clamp conditions. CCTs from mice kept under control conditions and fed a diet with normal levels of Na had very small [I.sub.NaS] (WT: 18 [+ or -] 13 pA; L/L: 22 [+ or -] 8 pA at [V.sub.m] = -100 mV) that were not different in WT and L/L animals. However, the L/L mice had much larger currents when the animals were fed a low-Na diet (WT: 256 [+ or -] 127 pA; L/L: 1,820 [+ or -] 330 pA) or infused with aldosterone (WT: 285 [+ or -] 63 pA; L/L: 1,600 [+ or -] 280 pA). Currents from L/L mice were also larger when animals were pretreated with a high-K diet but not when the CCTs were stimulated in vitro with 8-CTP-cAMP. Noise analysis of amiloride-induced fluctuations in [I.sub.Na] showed that single-channel currents at [V.sub.m] = 0 mV were slightly smaller in L/L mice (WT: 0.33 pA; L/L: 0.24 pA). This difference could be attributed to a decrease in driving force since current-voltage analysis indicated that intracellular Na was increased in the L/L animals. Analysis of spontaneous channel noise indicated that the open probability was similar in the two genotypes (WT: 0.77; L/L: 0.80). Thus the increase in whole cell current is attributed to a difference in the density of conducting channels. kidney; sodium transport; aldosterone; hypertension; amiloride

To test the role of epithelial Na channels in the day-to-day regulation of renal Na excretion, rats were infused via osmotic minipumps with the Na channel blocker amiloride at rates that achieved drug concentrations of 2-5 [micro]M in the lumen of the distal nephron. Daily Na excretion rates were unchanged, although amiloride-treated animals tended to excrete more Na in the afternoon and less in the late evening than controls. When the rats were given a low-Na diet, Na excretion rates were elevated in the amiloride-treated group within 4 h and remained higher than controls for at least 48 h. Adrenalectomized animals responded similarly to the low-Na diet. In contrast, rats infused with polythiazide at rates designed to inhibit NaCl transport in the distal tubule were able to conserve Na as well as did the controls. Injection of aldosterone (2 [micro]g/100 g body wt) decreased Na excretion in control animals after a l-h delay. This effect was largely abolished in amiloridetreated rats. On the basis of quantitative analysis of the results, we conclude that activation of amiloride-sensitive channels by mineralocorticoids accounts for 50-80% of the immediate natriuretic response of the kidney to a reduction in Na intake. Furthermore, the channels are necessary to achieve minimal rates of Na excretion during more chronic Na deprivation. amiloride; polythiazide; aldosterone; adrenalectomy

Inwardly rectifying K+ (Kir) channels act as a simple model system in the study of permeation and gating mechanisms. The first Kir channel, ROMK1 was cloned in 1993. In a study of ROMK2, single-channel kinetics were described by one open and two closed states. A new study indicates that the long-lived closed state represents block by a permeating divalent cation, while the short-lived closed state may be due to block by a permeating K+ ion.