A comprehensive guide to the ROMK potassium channel: form and function in health and disease

Abstract

The discovery of the renal outer medullary K⁺ channel (ROMK, K_ir1.1), the founding member of the inward-rectifying K⁺ channel (K_ir) family, by Ho and Hebert in 1993 revolutionized our understanding of potassium channel biology and renal potassium handling. Because of the central role that ROMK plays in the regulation of salt and potassium homeostasis, considerable efforts have been invested in understanding the underlying molecular mechanisms. Here we provide a comprehensive guide to ROMK, spanning from the physiology in the kidney to the organization and regulation by intracellular factors to the structural basis of its function at the atomic level.

the functional expression cloning of the renal outer medullary K⁺ channel (ROMK) by Ho and Hebert (50) revolutionized our understanding of renal potassium handling and opened an entirely new field of potassium channel biology. The discovery, together with the identification and molecular characterization of the bumetanide-sensitive Na⁺-K⁺-2Cl⁻ cotransporter NKCC2 (35), and the thiazide-sensitive Na⁺-Cl⁻ cotransporter NCC (36) in Hebert's laboratory, ushered in the molecular era of renal electrolyte transport. It also had a major impact outside of renal physiology and nephrology. As the defining member of a new potassium channel family, ROMK's identification revealed a channel architecture that differed from the typical voltage-dependent variety, shaping our current understanding of how potassium channel diversification is achieved. At the same time, the revelation of the ROMK molecular structure illustrated in no uncertain terms nature's conservation of the most basic potassium channel blueprint of potassium selectivity and conduction. Here we provide a comprehensive guide to ROMK, from its physiology in the kidney to an understanding of its function at the atomic level.

ROMK, the First Member of the K_ir Channel Family

With the discovery of ROMK, the identification of related channels rapidly followed, revealing an entirely new potassium channel family. Encoded by 15 different “KCNJ” genes, family members share a common biophysical property. All exhibit a nonlinear current-voltage relationship, characterized by a larger inward current than outward current, and are therefore commonly referred to as the inward-rectifying potassium channels or K_ir (63). They also share a common structure, distinguished by two transmembrane domains, a conserved potassium selectivity filter, and cytoplasmic NH₂- and COOH-terminal domains (Fig. 1). Functional K_ir channels are formed by homomeric or heteromeric assembly of four subunits. Although members of the K_ir channel family exhibit a number of common functional properties and a similar body plan, they are individually specialized for their pivotal roles in a wide range of physiological processes, including controlling membrane excitability, neuronal signaling, heart rate, vascular tone, hormone secretion, and epithelial salt transport. Importantly, defective K_ir forms are known to cause human disease, including inherited disorders of cardiac arrhythmias, hyperinsulinemic hypoglycemia, neonatal diabetes, and renal salt-wasting.

Fig. 1.

Renal outer medullary K⁺ channel (ROMK) isoforms. ROMK shares a common structure with other inward-rectifying K⁺ (K_ir) channels, typified by 2 transmembrane domains, a conserved potassium selectivity filter, and cytoplasmic NH₂- and COOH-terminal domain structures (A) and tetrameric assembly (B). ROMK1–3 isoforms (aka K_ir1.1a, -b, -c) have different NH₂-terminal structures (C) and are differently expressed along the nephron. DCT, distal convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct; cTAL, cortical thick ascending limb; OMCD, outer medullary collecting duct; mTAL, medullary thick ascending limb.

Function of ROMK in the Kidney

ROMK is the renal potassium secretory channel.

There should be little doubt that ROMK (aka K_ir1.1, product of the KCNJ1 gene) encodes the pore-forming subunit of the kidney's major potassium secretory channel (147) (Fig. 2). ROMK channels comprise the major apical membrane conductance in the thick ascending limb (TAL) and mediate the K⁺ efflux that is required by the Na⁺-K⁺-2Cl⁻ cotransporter NKCC2 for NaCl transport. They also contribute to the TAL transepithelial current flow and potential difference, important for paracellular Na⁺ and Ca²⁺ reabsorption. Under most conditions in cortical collecting duct (CCD) principal cells, these same channels represent the predominant and highly regulated K⁺ secretory pathway essential for K⁺ homeostasis (45). Patch-clamp studies of TAL cells and CCD principal cells previously characterized apical K_ir channels with distinctive biophysical, regulatory, and pharmacological properties, including weak inward rectification, high channel open probability (P_o), Ba²⁺ sensitivity, tetraethylammonium and quinidine insensitivity, and modulation by intracellular pH (pH_i), calcium, arachidonic acid, and vasopressin (10, 33, 144, 148–150). Moreover, these channels also exhibited inhibition by intracellular ATP and the sulfonylurea glibenclamide, activation by cAMP-dependent protein kinase, and inhibition by protein kinase C (64, 143, 145, 148).

Fig. 2.

ROMK encodes a major potassium secretory channel in the kidney. In distal nephron principal cells, ROMK1 and ROMK3 form the predominant 30-pS potassium channel. The high density and activity of these channels permit avid potassium flux into the tubular lumen. Together with the flow-dependent “BK” channel (shown in low-flow setting), ROMK channels are exquisitely regulated, adjusting potassium excretion to precisely match normal variations in dietary potassium intake. In thick ascending limb (TAL), potassium efflux through the ROMK2 channel is required to safeguard the efficient turnover of the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC). Potassium that is brought into the cell by NKCC2 recycles across the apical membrane through ROMK channels. In the TAL, ROMK2 encodes the 30-pS channel; the 70-pS channel is formed by ROMK2 and yet-to-be identified subunit(s). ENaC, epithelial Na⁺ channel; BLM, basolateral membrane.

Immunodetectable ROMK protein is found on the apical membrane and cytoplasmic compartments of the TAL, distal convoluted tubule (DCT), connecting tubule (CNT), and CCD (60, 93, 162), where these unique potassium secretory channels are expressed (33, 148, 149). Moreover, heterologous expression studies in Xenopus oocytes revealed that the biophysical and pharmacological properties of ROMK (∼35 pS, high P_o) are nearly indistinguishable from those of the predominant 35-pS secretory channel found in the TAL and CCD (50, 96, 102a). Finally, ROMK knockout studies in mice established a definitive link between ROMK and the renal secretory channel. Indeed, ablation of the ROMK gene (79) eliminates the 35-pS channel in the mouse TAL and CCD (82). Interestingly, the other TAL potassium secretory channel, which exhibits ROMK-like characteristics except for its higher single-channel conductance (70 pS) (150), is also lost in the ROMK-null mouse, making it likely that ROMK is also a critical subunit of the 70-pS K⁺ channel.

ROMK splice variants have different physiological roles.

Alternative splicing and promoter usage of the ROMK gene (located on human chromosome 11q24) generates three different products [ROMK1 (K_ir1.1a), ROMK2 (K_ir1.1b), ROMK3 (K_ir1.1c)] with different NH₂-terminal amino acid sequences (9, 127, 175) (Fig. 1C). Because ROMK1, ROMK2, and ROMK3 exhibit identical biophysical properties, their functional relevance is still not entirely clear. Nevertheless, they are differentially expressed along the nephron (Ref. 9; Fig. 1C) for different physiological duties (Fig. 2). ROMK2 channels in the TAL of Henle's loop are responsible for recycling potassium across the apical membrane to maintain avid NaCl reabsorption through the Na⁺-K⁺-2Cl⁻ cotransporter, important for the urinary concentrating mechanism (Fig. 2). ROMK1 and ROMK3 channels in the distal nephron constitute an important regulated component of the potassium secretory machinery of the kidney, essential for controlling renal potassium excretion and maintaining potassium balance (37).

Biophysical signature is well suited for secretion.

The biophysical properties of ROMK channels are well suited for their role in renal potassium secretion and recycling. Upon phosphorylation or ligand binding (see below), ROMK enters a long-lived, fully active conformation. Characterized by an open state (∼10 ms) that is momentarily interrupted by brief closures (∼1 ms) and longer, but less frequent, divalent cation blocking events (<1% events of ∼40 ms), active ROMK channels are wide open (P_o ∼0.9) (13). Furthermore, ROMK is weakly inward rectifying, permitting robust outward potassium flux at physiological membrane potentials. Together the two properties permit ROMK to function as a powerful conduit for avid potassium transport into the tubule lumen, important to maintain salt reabsorption in the TAL and to precisely match renal potassium excretion to dietary potassium intake.

A Multiprotein ROMK Complex

Association with ATP-binding cassette proteins.

Although ROMK shares the salient biophysical features of the small-conductance potassium channels in the TAL and collecting duct apical membrane, the absence of sensitivity to cytoplasmic adenosine triphosphate (ATP) suggested that the native channel might be more complex. Evidence from molecular reconstitution and gene knockout studies indicates that ROMK channels require an ATP-binding cassette (ABC) protein cofactor to manifest native channel properties. Coexpression of CFTR or the sulfonylurea receptor (SUR) (136) with ROMK in Xenopus oocytes leads to the formation of weakly inward-rectifying channels, which have acquired sensitivity to sulfonylurea agents (90) and ATP-dependent gating properties (119) just like the native channel (143, 145). In the kidney, CFTR plays a more important role than SUR. Indeed, expression patterns of ROMK and CFTR (19) overlap in the TAL and CCD apical membranes. Most importantly, Hebert, Lu, and coworkers (81) found that ATP and glibenclamide sensitivities of the 30-pS K⁺ channel in TAL and CCD cells are absent in CFTR-knockout mice, providing unequivocal evidence that the native ROMK secretory channel is regulated by CFTR. The concept has precedent, with an ever-growing body of data demonstrating that CFTR acts as a “conductance regulator,” in addition to its role as a Cl⁻ channel (126).

The requirement of CFTR to regulate ROMK function is similar to prototypical ATP-sensitive K⁺ (K_ATP) channels (3), which are comprised of the SUR and K_ir6 channel subunits (55). Although this may suggest that ATP-dependent regulation of potassium channels is generally rooted in the formation of ABC-K_ir channel complexes, several important aspects of ROMK-CFTR distinguish it from classic K_ATP channels (49, 81, 166). Unlike K_ir6-SUR channels, which assemble through direct interactions between the two subunits (125), interaction between ROMK and CFTR appears to be indirect and to require scaffolding proteins (166). Second, in contrast to the fixed and static interaction of K_ir6-SUR subunits, association of CFTR with ROMK appears to be dynamic and highly regulated. Indeed, protein kinase A (PKA)-mediated phosphorylation abrogates the functional interaction between CFTR and ROMK, releasing the channel from ATP-dependent inhibition (81). Finally, the requirements for proper trafficking of ROMK channels to the plasma membrane are much different from the archetypical K_ATP channels. SUR and K_ir6.x subunits contain endoplasmic reticulum (ER) retention/retrieval signals that are masked upon full assembly of the K_ATP channel complex (172). By contrast, ROMK exit from the ER is not facilitated by CFTR (166). Instead, scaffolding proteins and a phosphorylation-dependent mechanism are involved (see below).

Scaffold-dependent assembly of a multiprotein complex.

Recruitment of regulatory factors to the ROMK channel can be orchestrated by A-kinase anchor proteins (AKAPs) (1, 2) and PDZ proteins (142, 166, 168). The Na⁺/H⁺ exchange regulatory factor NHERF-2, which colocalizes with ROMK in the rat collecting duct (142), has been the focus of attention. Containing tandem PDZ domains and an ezrin/radixin/mosein/merlin (ERM) binding domain, NHERF-2 has the capacity to act as both an AKAP and a PDZ scaffold (155). ROMK preferentially interacts with the first NHERF-2 PDZ domain (166), allowing the other PDZ domain and the ERM domain to recruit regulatory factors to the channel. The ERM binding domain engages the actin-binding AKAP ezrin, likely to juxtapose PKA with the channel for efficient phosphorylation (154). CFTR (135) and serine- and glucocorticoid-regulated kinase (SGK)-1 (100) both interact with NHERF-2 via the second PDZ domain. Consequently, NHERF-2 facilitates the assembly of ternary complexes containing ROMK and CFTR or SGK-1 for optimal channel regulation.

Regulation of ROMK

Regulation of gating.

Factors that influence the activity of ROMK channels in the TAL and collecting duct have profound effects on the renal concentrating mechanism and potassium excretion. A growing body of evidence indicates that ROMK channels are activated and maintained in a high-P_o state by the concerted actions of PKA phosphorylation, phosphatidylinositol 4,5-bisphosphate (PIP₂) interaction, ATP binding, and pH_i.

PROTEIN KINASE A.

Like the native secretory channel (146), ROMK activity is dependent on PKA phosphorylation (92, 163). In fact, activation of ROMK by PKA is thought to underlie the regulation of renal potassium transport by vasopressin (10). Biochemical approaches identified three separate PKA phosphoacceptor sites within the cytoplasmic NH₂ (S₄₄) and COOH termini (S₂₁₉ and S₃₁₃) (163). All must be phosphorylated for full channel function (88). Phosphorylation of the NH₂-terminal residue, S₄₄, is absolutely required for cell surface expression (99, 165, 167). Phosphorylation of the two COOH-terminal sites, by contrast, maintain the channel in a high-P_o state (88). Present evidence indicates that phosphorylation of these residues controls the channel opening process by modulating cytoplasmic ligand-dependent gating of the channel [pH (69), ATP (81) or PIP₂ (75)].

PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE.

With the discovery of ROMK and other members of the K_ir channel family, a completely unappreciated and novel mode of channel regulation emerged. After a report that generation of PIP₂ by ATP-dependent lipid kinases activates K_ATP channels in the heart (48), Huang et al. (52) explored the mechanism in several recombinant K_ir channels. Remarkably, they found that PIP₂ directly binds to ROMK, and this is absolutely necessary for channel opening. The seminal observation provoked a torrent of intense research activity, revealing the central role that PIP₂ plays in the regulation of a wide range of ion channels and ligand-gated receptors (51).

It is now clear that PIP₂ is an essential K_ir channel cofactor, required to maintain the open state of all known inward-rectifying potassium channels. Because the apparent PIP₂ binding affinity varies widely among different K_ir channels, different K_ir subtypes differ in the way PIP₂ regulates them. ROMK tightly interacts with PIP₂ (52). Consequently, it is not as susceptible to physiological changes in PIP₂ levels as other K_ir channels (25). Nevertheless, activation of PIP₂ hydrolysis by PKC inhibits ROMK channel activity in Xenopus oocytes and was suggested as a mechanism (170), among others (132), to explain how PKC modulates ROMK channels in the collecting duct (146). Formation of PIP₂ through membrane-bound phosphoinositide kinases is required to maintain ROMK activity in vivo (80), but it remains to be determined whether ROMK activity in the kidney is controlled by physiological modulation of PIP₂ levels.

ATP.

ROMK is not a classic K_ATP channel like those found in pancreatic islet cells, but similar to the potassium secretory channels in the TAL and CCD (145), it is inhibited by cytoplasmic ATP so long as it is expressed with CFTR (81, 119). Because ATP-dependent modulation occurs in the physiological range of ATP levels (K_1/2 ∼1 mM) and is relieved by ADP, ROMK activity is likely to be directly coupled to the activity of Na⁺-K⁺-ATPase (156). Consider, for example, the sequence of cellular events that might accompany an elevation of plasma potassium after ingestion of a potassium-rich meal. Although measurements still need to be made, stimulation of Na⁺-K⁺-ATPase activity by the substrate action of potassium (94) could conceivably lower the ratio of intracellular ATP to ADP and thereby release ROMK from inhibition to augment potassium excretion. Furthermore, because PKA abrogates the functional interaction between CFTR and ROMK, Hebert and coworkers (81) proposed that the distribution of open and ATP-inhibited ROMK channels in apical membranes may be regulated by hormones to modulate potassium secretion in concert with homeostatic demands.

INTRACELLULAR PH.

Like the native channel, ROMK channel activity is exquisitely sensitive to pH_i. Cytoplasmic acidification inhibits ROMK, inducing a long-lived closed state (17, 91). The reduction in channel P_o is highly cooperative and occurs with an apparent acidic dissociation constant (pK_a) near neutral pH_i (17, 27, 91), making channel activity especially susceptible to pathophysiological changes in pH_i. Perhaps more interestingly, the apparent pK_a of the channel is physiologically controlled. In fact, accumulating evidence indicates that PKA (69)- and PIP₂ (72)-dependent activation of ROMK work in an interdependent fashion with the pH-dependent gating mechanism. Studies of Leipziger et al. (69) demonstrating that pH sensitivity is dynamically controlled by phosphorylation provide a salient example. These investigators demonstrated that PKA phosphorylation of the COOH-terminal residues, S₂₁₉ and S₃₁₃, causes an acid shift in the apparent pK_a, and this activates the channel at physiological pH_i. Just how the chemical modification of sites within the large cytoplasmic COOH terminus is translated to the channel pH_i-gating process has been the subject of great interest. As discussed below, insights from recently solved atomic resolution structures of related inward rectifier channels provide a plausible mechanism (see below).

Regulation of ROMK Surface Density

Because ROMK channels normally exhibit a very high P_o, physiological modulation of channel activity, as controlled by hormones and dietary potassium, is largely achieved by regulated changes in the number of active channels on the plasmalemma. In addition to gating mechanisms that switch on and off resident channels at the apical surface, membrane trafficking mechanisms play an important role. Regulated movement of ROMK to and from the apical surface is particularly important in the distal segments of the nephron, where apical channel expression appears to be precisely regulated, adjusting potassium excretion to exactly match dietary intake. In recent years, the molecular mechanisms have begun to be elucidated.

Forward trafficking in the secretory pathway.

Adaptive changes in the distal nephron principal cell take place in response to an increase in dietary potassium, allowing a more effective and enhanced excretion of potassium (37). The response, called potassium adaptation, involves an increase in the number of active ROMK-type channels on the apical surface (102, 148). Although the traditional text book view holds that aldosterone plays a direct role, it does not appear to be sufficient (104). The mediators of the response still remain unsettled, but other adrenal hormones (102), plasma potassium itself, and other unidentified kaliuretic factors (reviewed in Ref. 103) have been implicated. Nevertheless, the boost in channel density is rapid (101) and occurs without changes in ROMK transcript abundance (34), consistent with a posttranslational mechanism. Recent studies implicate a phosphorylation-dependent trafficking process.

Indeed, cell surface expression of ROMK requires direct phosphorylation (167). A putative ER localization signal in the extreme cytoplasmic COOH terminus of ROMK normally prevents channels from reaching the plasmalemma (99, 165), that is, until phosphorylation of a cytoplasmic NH₂-terminal residue, S₄₄, creates a separate trafficking structure that effectively overrides ER retention and drives anterograde movement of the channel through the secretory pathway. Importantly, the phosphorylation-dependent trafficking site in ROMK (a R₃₉XRXXSp motif) exhibits sequence similarity with a comparable NH₂-terminal structure in the K_ir2.1 channel (R₄₄XR) that is required for constitutive traffic out of the Golgi (134), suggesting that the NH₂-terminal structure in ROMK serves a related Golgi export function. The observations contribute to a new and evolving view that the cell surface density and composition of certain types of channels and receptors can be adjusted by controlling their export from the ER and Golgi (85). ROMK should prove to be an ideal model to explore the mechanisms. It will be critical to determine whether the phosphorylated structure acts as an autonomous Golgi export signal and to identify the intracellular trafficking machinery that decodes it.

Observations that phosphorylation of S₄₄ is mediated by either PKA or the aldosterone-induced kinase SGK-1 imply that the phosphorylation-dependent trafficking process plays an important role in the upregulation of ROMK channel density by vasopressin and dietary potassium. Consistent with a central function in potassium adaptation, a rapid increase in SGK-1 is observed in the kidney after the ingestion of a large dietary potassium load (167), coincident with the early increase in ROMK channel density (104). Renal potassium excretion is impaired in SGK-1-null mice, but further studies are required to critically determine the subcellular compartment in the CCD where ROMK channels accumulate (54). Because SGK-1 must be activated by phosphorylation after it is induced by steroid hormones, it is well poised to integrate the effects of adrenal hormones and suspected kaliuretic signals (103) for an appropriate potassium adaptation response.

Endocytosis.

Membrane trafficking processes also underpin the physiological downregulation of ROMK channels. Present evidence indicates that channels are retrieved from the apical surface by clathrin-dependent endocytosis to limit urinary potassium loss in states of dietary potassium deficiency (18, 133, 169). Investigation in this area has largely centered on the underlying signal transduction mechanisms, highlighting Src (73, 74, 133) and the WNK kinases (58, 68, 70, 141).

SRC.

Members of the protein tyrosine kinase (PTK) directly phosphorylate ROMK1 at position Y₃₃₇ and reduce channel surface expression through a dynamin-dependent (74) and concanavalin A-sensitive (152) process, suggesting a phosphotyrosine-dependent endocytotic mechanism. Significantly, dietary potassium restriction is accompanied by an increase in kidney cSrc and c-Yes (153) and a parallel rise in Y₃₃₇ phosphorylation (74). Pharmacological studies suggest that attenuation of protein tyrosine phosphatases (133) and/or activation of PTK are necessary for physiological suppression of ROMK (153). Therefore, it seems likely that phosphorylation of tyrosine residue 337 is required for ROMK1 internalization in dietary potassium depletion.

WNK KINASES.

Since the discovery that mutations in the WNK1 and WNK4 kinase genes cause pseudohypoaldosteronism type II, a familial disorder of renal potassium retention and hypertension (158), there has been great interest in understanding how the WNKs normally control the balance between renal potassium excretion and sodium reabsorption. Although concepts about WNK signaling in the kidney are somewhat controversial and in constant flux (53), present evidence strongly suggests that WNKs control salt balance through the divergent modulation of the Na⁺-Cl⁻ cotransporter NCC and the ROMK channel (57).

Work in heterologous expression systems indicates that WNK1 (68, 141), WNK3 (70), and WNK4 (58) downregulate ROMK at the cell surface by stimulating clathrin-dependent endocytosis. Similar to the endocytotic signals found in LDL receptors, the ROMK channel contains an “NPX”-type motif that is necessary for its internalization (169). Mutation in the key asparagine residue not only impairs ROMK endocytosis (169), it also renders the channels resistant to WNK kinases (58, 68, 141). A trafficking scaffold, intersectin, recruits WNK kinases to clathrin-coated pits (41), but it is still not clear how ROMK channels are similarly targeted to sites of endocytic retrieval.

The abundance and activity of WNK kinases are tightly controlled in the kidney, apparently to regulate ROMK endocytosis in concert with physiological demands. For example, WNK4 loses its ability to inhibit ROMK when it is phosphorylated by the aldosterone-induced kinase SGK-1 (116), providing an additional mechanism to enhance ROMK surface expression with dietary potassium loading. Two WNK1 isoforms (the long form, L-WNK1 and the kidney specific form, KS-WNK1) are also regulated by dietary potassium. Unlike L-WNK1, the kinase-deficient KS-WNK1 form has no inhibitory effect on ROMK (68, 141). Instead, KS-WNK1 negatively modulates L-WNK1 to suppress channel endocytosis (68, 141) and enhance the apical surface expression of ROMK in the distal nephron (76). Acute dietary potassium loading increases the relative abundance of KS-WNK1 (141), while dietary potassium restriction increases the relative abundance of L-WNK1, suggesting that a WNK1 isoform switch plays an important role in the physiological regulation of ROMK apical surface density.

ROMK in Disease

Bartter's syndrome.

A central function of ROMK in human health and disease was illuminated by discovery of the genetic basis of Bartter's syndrome (5), a group of rare autosomal recessive salt-wasting nephropathies characterized by polyuria, hypokalemia, metabolic alkalosis, and hypotension. Bartter's disorders have diverse genetic origins, but they share a common pathological mechanism, namely, a severe reduction in salt reabsorption by the TAL (42). Indeed, the disease is caused by loss-of-function mutations in any one of the four major components of the NaCl reabsorptive machinery in the TAL or by gain-of-function mutations in the calcium-sensing receptor (type V) (139, 151), which normally negatively regulates sodium reabsorption in the TAL (43). Type I Bartter's disease involves the Na⁺-K⁺-2Cl⁻ cotransporter NKCC2 (130). Type II disease is caused by inactivating mutations in ROMK (59, 128). Type III involves the basolateral Cl⁻ channel ClCKb (61, 129). In type IV, the accessory subunit of ClCkb, Barttin, is affected (8).

Pioneering physiological studies of Greger and Schlatter (38) and Hebert, Freidman and Andreoli (44, 46) in the isolated, perfused TAL strongly suggested that apical potassium channels were required to constantly supply potassium into the tubule lumen to maintain the turnover of the Na⁺-K⁺-2Cl⁻ cotransporter for NaCl reabsorption. The discovery that loss-of-function mutations in ROMK cause Bartter's syndrome, together with subsequent studies in ROMK-knockout mice (4, 79, 82), provided airtight genetic evidence of the transport mechanism and firmly established the molecular identity of the apical potassium channels in the TAL as ROMK.

The discovery also posed a riddle. That is, if ROMK channels in the distal nephron play a central role in potassium excretion, how can potassium wasting in Bartter's syndrome be explained? Work to address the conundrum revealed that two independent potassium secretory mechanisms rather than just one usually maintain potassium excretion. The seminal discovery that “big potassium” (BK) channels are required for flow-dependent potassium secretion (111, 115, 159) illuminated the ROMK-independent potassium secretory pathway. Bailey et al. (4) found that potassium secretion in ROMK-null mice is maintained by upregulation of a flow-dependent, iberiotoxin-sensitive pathway, establishing that compensatory overexpression of BK channels permits potassium excretion in the high-flow setting of Bartter's syndrome. In the same way, upregulation of ROMK channels preserves potassium secretion in BK channel-knockout animals (115). It would seem that the kidney has evolved separate ROMK- and BK-mediated potassium secretory mechanisms to ensure high-capacity potassium excretion and to safeguard against fatal hyperkalemia.

The clinical phenotype that is manifested in patients with inactivating mutations in ROMK is somewhat different from that observed with other forms of Bartter's syndrome (108). This is likely a consequence of the dual role that ROMK normally plays in the healthy kidney, both controlling NaCl reabsorption in the TAL and regulating distal potassium secretion. For example, potassium wasting and hypokalemia in type II Bartter's syndrome is relatively mild compared with other forms of the disease as a consequence of the diminished capacity to excrete potassium. Interestingly, newborn infants with type II Bartter's syndrome actually exhibit a transient period of urinary potassium retention and hyperkalemia before becoming normo- to hypokalemic later in life (29). Neonatal type II patients may be particularly vulnerable to potassium overload because development of BK-dependent potassium secretion is delayed (122, 160).

Over 35 different Bartter's disease mutations have been identified in ROMK. Several introduce nonsense codons or frameshifts in the NH₂ terminus, producing truncated proteins with obvious loss-of-function consequences (59, 128). Over half of the missense mutations reduce or even eliminate surface expression of ROMK (107), presumably as a result of global misfolding or mistrafficking. Others alter channel opening by disrupting the potassium permeation pathway or by upsetting the way the channel is regulated by phosphorylation, PIP₂ binding, or pH_i. As discussed below, much has been learned about the structural basis of channel function by exploring how the disease-causing mutations alter channel gating.

Structure-Function Relationships

Atomic resolution structures of closely related inward-rectifying potassium channels have recently been solved (65, 97, 98, 106), revealing a common K_ir body plan (Fig. 3). A unique potassium conduction pathway, involving the canonical transmembrane pore and a central cavity of a large cytoplasmic domain, characterizes inward-rectifying potassium channels. The distinctive architecture likely accounts for their specialized properties of potassium conduction, inward rectification, and ligand-dependent gating. Here we review and interpret extensive mutagenesis studies in the context of a homology-based atomic model of ROMK structure (Fig. 3) to best appreciate the structural basis of ROMK function in health and dysfunction in disease.

Fig. 3.

An atomic model of ROMK was developed with known K_ir channel structures and an iterative optimization algorithm (28). The major body of ROMK is depicted in 2 subunits (amino acids 34–356 of ROMK1 are shown; residues 60–64 and 185–191 are not resolved). Images were rendered with PyMol software. TM1, TM2, first and second transmembrane domains.

Transmembrane potassium conduction pathway.

POTASSIUM SELECTIVITY.

Four ROMK subunits assemble around a central pore that crosses the plasmalemma. Linkers connecting the two transmembrane domains project from the extracellular space into the membrane like staves in a barrel, forming the narrowest part of the open pore conduction pathway. This structure, called the P loop, contains the conserved potassium selectivity sequence (47). Identified by a “T[V/I]GYG” motif, the sequence adopts a strand conformation in which backbone carbonyl oxygens point into the pore. Like all known potassium channels, the close juxtaposition of identical structures from each ROMK subunit creates four equally spaced binding sites for potassium (Fig. 4). Four oxygen atoms at each binding site effectively “cage” a potassium ion, similar to the way water molecules surround potassium in free solution. By mimicking the inner hydration shell of potassium, the selectivity filter creates energetically favorable sites for potassium to diffuse from the aqueous environment. Because the binding sites do not counterbalance dehydration of smaller ions, like sodium, selectivity is restricted to potassium (89). The potassium selectivity of ROMK is remarkably high, P_K/Na > 20–100 (P_Tl > P_K > P_Rb > P_NH4) (12, 175). Of note, a Bartter mutation, I₁₄₂T, in the second residue of the selectivity sequence disrupts potassium permeation (107).

Fig. 4.

ROMK potassium selectivity filter. The selectivity motif “T₁₄₁IGYG” adopts a strand conformation in which backbone carbonyl oxygens (red atoms) point into the pore, mimicking the hydration shell of potassium. V₁₄₀, located between the pore helix and the selectivity filter, is a major determinant of the ROMK single-channel conductance and barium block. Rapid conformational movement of T₁₄₁I₁₄₂ likely underpins the fast gating process of ROMK. Residues in red are mutated in Bartter's syndrome type II.

POTASSIUM CONDUCTION.

As inferred from early biophysical measurements (83), potassium moves through the ROMK pore by means of a multiple ion conduction mechanism. Viewed in the archetypal KcsA channel, pairs of potassium ions appear to simultaneously occupy different binding sites at the selectivity filter, depending on its conformation (118). Rapid cycles of potassium entry, binding, and conformational change, together with electrostatic repulsion of potassium ions (89), allow potassium to move through the pore with extraordinary throughput (∼10⁸ K⁺ ions/s) and remarkable selectivity (6, 89).

As a consequence of potassium binding at the selectivity filter, the P-loop region is a major determinant of the ROMK single-channel conductance (16). Although the selectivity filter is highly conserved, rates of potassium binding, release, and passage likely vary among different potassium channel types, giving rise to differences in their single-channel conductances. In K_ir channels, the P-loop residues that are responsible for maintaining the structure and flexibility of the selectivity filter are thought to influence this. Like other K_ir channels, the ROMK P loop contains a highly conserved glutamate-arginine residue pair (E₁₃₆-R₁₄₇), which maintains the rigid bowlike structure of the selectivity filter (20, 164). Neighboring nonconserved residues flanking the salt bridge likely affect the ROMK signature single-channel conductance. In particular, V₁₄₀, located between the pore helix and the selectivity filter (Fig. 4), contribute to differences in the single-channel conductance between ROMK and its close cousin, K_ir2.1 (174). V₁₄₀ also influences cation selectivity and barium block.

FAST GATING.

ROMK exhibits a fast gating process, characterized by rapid transitions between the open (∼10 ms) and shortest-lived closed (∼1 ms) states (13). Because the rate of entering the short-lived closed state varies with the K⁺ concentration and is proportional to ROMK current amplitude, it was suspected that K⁺ permeation plays a key role (13–15). In fact, recent molecular simulation studies reveal that potassium occupancy in the pore has a profound influence on the structure of the selectivity filter. Cation occupancy at a particular position in the selectivity filter causes a conformational change at the position equivalent to T₁₄₁-I₁₄₂ in ROMK (Fig. 4), which briefly shuttles the channel into a nonconducting conformation (7, 21). The event occurs in the same time frame as a fast closure, consistent with a mechanism of fast gating. Alternatively, variable energy of potassium binding in the pore has been proposed, in which a short-lived state binds potassium so tightly that it briefly plugs the permeation pathway (15).

INWARD RECTIFICATION.

Intracellular Mg²⁺ (84, 96) and polyamines (77) enter the K_ir channel cytoplasmic pore at depolarizing potentials, bind to a site near the selectivity filter, and plug the potassium permeation pathway (67), giving rise to inward rectification. Because of subtle differences in the chemistry of the binding site, the affinity for Mg²⁺ and polyamines as well as the degree of inward rectification vary widely among different members of the K_ir channel family. A single amino acid within the second transmembrane domain (TM2), corresponding to N₁₇₁ in ROMK, largely accounts for the rectification phenotype. A mutation, N₁₇₁D, in ROMK produces strong inward rectification, whereas the reverse mutation, D₁₇₂N, in the strong inward-rectifying channel K_ir2.1 produces ROMK-like rectification (157). Consistent with a binding site in the transmembrane potassium conduction pathway, the side chain of N₁₇₁ projects from TM2 into the pore (Fig. 5).

Fig. 5.

The ROMK transmembrane potassium conduction pathway contains the key determinants of inward rectification (N₁₇₁) and regulated channel gating. Gating movement is thought to involve motion about the glycine hinges, pulling L₁₇₉, the putative gate, out of the pore. Hydrogen bonding between K₈₀, at the base of TM1, and A₁₇₇, on TM2, stabilizes the gate. Y₇₉ is mutated in Bartter's syndrome.

REGULATED GATING.

The transmembrane conduction pathway is also thought to contain the regulated gate that opens in response to cytoplasmic alkalization, ATP dissociation, or PIP₂ interaction. The subject of K_ir channel gating mechanisms is not without controversy, and much remains to be explored (26, 109, 161). Nevertheless, it is widely believed that regulated channel opening involves the movement of the pore helixes (TM2) away from the so-called “bundle crossing.” Structural homology modeling and extensive mutagenesis studies in ROMK are consistent with this idea. Glycine residues (G₁₆₇ and G₁₇₆; see Fig. 5) have been implicated as the flexible “hinges” that allow the TM2 helixes to pivot away from one another (121). The movement would carry the putative gate at the cytoplasmic end of TM2 away from the pore. As shown in Fig. 5, the hydrophobic alkane side chain of L₁₇₉ projects from the base of TM2 into the closed pore. Replacement of this residue with small or highly polar side chains stabilizes the open state (95, 120), just as would be predicted if L₁₇₉ acts as the regulated gate, occluding the conduction pathway in the closed state.

Interactions between the two transmembrane helixes also influence the regulated gating process (Fig. 5). Strong evidence from molecular modeling and mutagenesis studies suggests that hydrogen bonding between the backbone carbonyl oxygen of A₁₇₇, and the ε-nitrogen of K₈₀ on the first transmembrane domain (TM1), controls the energetics of the PIP₂- and pH-dependent gating process (113, 114). A Bartter mutation that replaces A₁₇₇ with threonine (107) disrupts the interaction and causes a profound alkaline shift in the pH sensitivity of ROMK (114), effectively turning off channel function at physiological pH_i. Substitution of K₈₀ with residues that are incapable of H-bonding with A₁₇₇ alters gating in a similar way (113). A Bartter mutation, involving a nearby lipid-facing residue, Y₇₉, at the base of TM1, also disrupts regulated channel opening (107). Interestingly, mutations in the comparable residues of the K_ir6.2 K_ATP channel alter ATP-dependent gating and produce neonatal diabetes (39). In light of these observations, it seems probable that the energetics of regulated K⁺ channel opening are strongly influenced by the H-bonding interactions at the base of TM1 and TM2 and the interaction of TM1 with the inner lipid leaflet.

Cytoplasmic Domain

Extended pore.

The cytoplasmic regions of ROMK assemble (32, 62) to form a long water-filled pore, extending ∼30 Å from the canonical transmembrane potassium conduction pathway (Fig. 6). An antiparallel arrangement of COOH-terminal β-strands lines the inner cavity of the cytoplasmic pore with acidic residues. Electrostatic free energy calculations indicate that this architecture creates a favorable environment for efficient potassium transport and cation blocker binding (117). Remarkably, there is a high degree of concordance between residues that contributes to the favorable static field and residues known from mutagenesis studies to affect cation block and single-channel conductance. For example, acidic amino acids in the ROMK cytoplasmic pore (D₂₅₄, E₂₅₈, D₂₉₈) largely correspond to conserved residues that control the rate of polyamine entry (66, 67). Other residues in the inner wall directly influence the transport of potassium through the cytoplasmic pore. These cytoplasmic structures act as energy barriers to K⁺ transport and, consequently, affect the single-channel conductance independently of structures in the transmembrane selectivity filter (16). N₂₅₉ plays the most significant role (173). Importantly, the N₂₅₉ side chain lines the inner wall of the pore and contributes to surface charge of this structure (117).

Fig. 6.

The cytoplasmic domains of ROMK. Two subunits are shown. Top: standard cartoon view. Bottom: surface rendering. Pore lining acidic residues are shown in navy blue. N₂₅₉ (light blue) plays an important role in determining the single-channel conductance. The G loops (magenta) at the apex of the cytoplasmic domain likely participate in gating. Phosphatidylinositol 4,5-bisphosphate (PIP₂) binding residues are shown in green. Putative ATP binding domain is shown in yellow (bottom). Note proximity of putative ATP binding residues K₁₉₆ and G₃₅₅. Residues mutated in Bartter's syndrome are shown in red.

Gating structure in the cytoplasmic pore.

A narrow opening at the apex of the cytoplasmic pore is formed by the coalescence of four identical loops that project from the COOH-terminal βC- and βD-strands of each subunit (Fig. 6). This structure, called the G loop, is believed to create a flexible diffusion barrier between the cytoplasmic and transmembrane pore. First recognized in K_ir2.1 and K_ir3.1, mutations in G-loop residues, which face into the central axis of the cytoplasmic pore (corresponding to T₃₀₀ in ROMK1), alter channel gating and inward rectification (106). Analysis of K_ir2.1-ROMK chimeras revealed that a comparable structure (residues 294–320) in ROMK contributes to the single-channel conductance, consistent with the notion that the G loop acts as a resistance barrier to ion flow in ROMK (173). Importantly, a Bartter mutation, A₃₀₆T, that disrupts potassium conduction (107) is located in the G loop. Disease-causing mutations in other K_ir channels also affect the G loop, underscoring the general importance of the structure in the inward rectifiers (86, 105).

In fact, the G loop has been implicated in regulated K_ir channel gating. In K_ir channel crystal structures, the G loops adopt two different conformations, depending on rigid body movements of the cytoplasmic domains (97, 106). In one state, the G loops are wide enough apart to permit the passage of a partially hydrated potassium. In the other, the G loops fold into a constricted conformation and pitch off the conduction pathway, making it too small to allow transit of even a dehydrated potassium. Present evidence is consistent with the idea that these conformational changes are physiologically relevant. Indeed, state-dependent chemical modification studies in ROMK detected a conformational movement of a G-loop residue, C₃₀₈, during the pH-dependent gating process (124). Because mutagenesis and electrophysiological studies with K_ir2.1 (86, 106), K_ir3.2 (30, 40, 56), and K_ir6.2 (112) independently mapped residues in or near the G loop as critical determinants of ligand-dependent gating, it seems likely that the G loop plays a fundamental role in a general K_ir channel gating mechanism.

Given the physical proximity of the TM2 bundle crossing to the G loop (Fig. 3), it has been proposed that ligand binding (i.e., PIP₂) or phosphorylation at nearby sites on the surface of the cytoplasmic domain is allosterically coupled to TM2 movement through conformational changes at the G loop (86, 97, 106). Disruption of the coupling mechanism has been proposed to explain disease-causing gating defects in K_ir2.1 and K_ir6.2, involving mutations in G-loop residues (86, 105, 112). Obviously, further studies are required to critically test these ideas in ROMK.

Channel modulation.

The large cytoplasmic domain structure has an enormous solvent-exposed surface area, important for docking modulators for ligand (PIP₂, pH, ATP) and phosphorylation-dependent gating.

PIP₂.

Systematic mutagenesis revealed a group of positively charged cytoplasmic amino acid residues (corresponding to R₄₈, K₁₈₁, K₁₈₄, K₁₈₆, R₁₈₈, R₂₁₇, K₂₁₈, R₃₁₁ in ROMK1) that are required for normal PIP₂-dependent gating in ROMK and other K_ir channels (78, 171). Mutagenic neutralization of these residues reduces channel activity and speeds up the rate of inactivation that occurs upon exposure to anti-PIP₂ antibodies or polycationic chelating agents. Because the negatively charged head group of PIP₂ interacts with positively charged surfaces, identification of these residues points to a conserved PIP₂ interaction site. Consistent with a binding site rather than an allosteric modulator, MacGregor et al. reported (87) that a COOH-terminal ROMK peptide (amino acid residues 183–221, containing K₁₈₁, K₁₈₄, K₁₈₆ and R₁₈₈), has the capacity to directly bind PIP₂. Much of the segment is not resolved in K_ir crystal structures but corresponds to the initial COOH terminus that extends from TM2 and is likely to reside at the apex of the cytoplasmic domain. Mapping the other basic residues in the three-dimensional (3D) model reveals most (R₄₈, R₂₁₇, R₂₁₈) cluster at the top of the cytoplasmic domain (Fig. 5), where they are predicted to face the inner lipid leaflet. Collectively, the observations indicate that PIP₂ directly binds to the apical surface of the cytoplasmic domain, providing rationale for the working hypothesis that PIP₂ maintains the channel in an open state by tethering the extended cytoplasmic pore to the plasma membrane.

It is noteworthy that a group of Bartter mutations (C₄₉Y, I₅₁T, A₂₁₄V, and L₂₂₀F) disrupt PIP₂-dependent gating (78). Mapping the residues on the 3D ROMK model reveals that they cluster on the membrane-facing surface of the cytoplasmic domain near the putative PIP₂ binding surface, making it likely that the mutations alter the PIP₂ binding pocket. Similar observations with disease-causing mutations in K_ir2.1 (Andersen-Tawil, Ref. 110) have been interpreted to indicate that a decrease in channel-PIP₂ interactions underlies many K_ir channelopathies (78).

ATP.

In a series of elegant studies, Steve Hebert's group demonstrated that ATP directly interacts with ROMK. Measurements of fluorescent ATP analog binding to purified, recombinant bacterial fusion proteins of the ROMK cytoplasmic domain revealed that the first 39 COOH-terminal amino acid residues of ROMK1 (183–221) are sufficient to support high-affinity ATP binding (138). Three arginines (R₁₈₈, R₂₀₃, and R₂₁₇) in this segment, known to be important for modulating ATP gating, were subsequently found to be essential for the interaction (22). Because these residues also comprise part of the PIP₂ interaction site, competitive binding of the two ligands at the same locale (87) offers a likely explanation for the antagonistic regulation of ROMK by PIP₂ and ATP.

High-affinity ATP binding is also governed by K₁₉₆ and R₂₁₂ and two distal segments of the ROMK COOH terminus (G₃₃₅KY and F₃₄₄GKTVE) (23). Among the K_ir channel family, the structures exhibit an especially high degree of similarity to classic K_ATP (K_ir6.x) channels. Extensive mutagenesis and functional studies indicate that the corresponding structures in K_ir6.2 K_ATP [particularly those analogous to K₁₉₆ (137) and G₃₃₅ (24) in ROMK] govern ATP dependent gating. Remarkably, these residues are in close proximity in the 3D structure, forming a common patch on the cytoplasmic domain surface of each subunit, consistent with a novel nucleotide-binding domain (see Fig. 5).

PH.

Identifying the structures that determine ROMK modulation by pH_i has been the focus of intense investigation and the subject of some controversy. Careful and convincing studies provided multiple independent lines of evidence that lysine at position 80 acts as the pH sensor in ROMK (27). Changing K₈₀ to methionine dramatically reduced the pH sensitivity of ROMK channels; the reverse mutation (M₈₄K) converted a pH_i-resistant channel, K_ir2.1 (pK_a ≤5), to a highly pH-sensitive one (pK_a ∼7). Chemical modification studies indicated that K₈₀ is accessible to the cytoplasm (27). Furthermore, 9-flurenylmethylchloroformate, a reagent that reacts with unprotonated NH₂ groups on lysines to prevent proton attack, renders ROMK largely insensitive to cytoplasmic acidification (123).

With the identification of two cytoplasmic arginine residues, R₄₁ and R₃₁₁, that are required for setting the apparent pK_a near neutral pH, a mechanism for the anomalous titration of K₈₀ was put forward (123). According to this model, the pK_a of K₈₀, which would be near pH 11 if it were in free solution, is brought into the physiological range by electrostatic shielding of R₄₁ and R₃₁₁. The mechanism implied a ROMK structure in which K₈₀, R₄₁, and R₃₁₁ were in close physical proximity (123).

The RKR triad concept quickly fell out of favor when the atomic structures of related channels were solved and ROMK modeling revealed that R₄₁ and R₃₁₁ are nowhere near K₈₀, being over 24 Å apart (114). In the 3D model, K₈₀ is located at the cytoplasmic tip of TM1 (Fig. 5); R₄₁ and R₃₁₁ are embedded deep within the cytoplasmic domain, where they form highly conserved intrasubunit (R₄₁-E₃₁₈) and intersubunit (R₃₁₁-E₃₀₂) salt bridges (71, 114). Importantly, bridge-disrupting mutations, including R₃₁₁W/Q in Bartter's disease (107, 123), increase pH_i sensitivity and cause channel closure at physiological pH (71, 114), underscoring the importance of the interactions in channel gating. Further investigation is required to elucidate the mechanism, particularly to explore whether these interactions control the conformation of the pH sensor or sensitize the pH-dependent gate.

The role of K₈₀ as the titratable pH sensor has also been debated. Arguments against the idea largely stem from observations with zebrafish and pufferfish orthologs of mammalian ROMK channels (114). Because the fish channels contain an isoleucine or valine at the equivalent position to K₈₀ yet exhibit normal pH gating in the physiological pH range, it has been suggested that the pH sensor must be located elsewhere, such as in the cytoplasmic domain (114). Others have reported that cytoplasmic histidine residues in mammalian ROMK channels (cytoplasmic pore residues H₂₂₅ and H₂₇₄ and the solvent-exposed surface residue H₃₅₄) might collectively act as the pH sensor (11); however, these are not conserved in the zebrafish and pufferfish orthologs, either. According to this view, the locus of the pH sensor in mammalian still remains unknown.

In our view, however, the jury is still out on the role of K₈₀. It is hard to completely dismiss all early studies, which so convincingly supported the pH sensor mechanism. Furthermore, the low homology of fish forms to mammalian ROMK channel (<60% amino acid identity) makes it difficult to make inferences about the functional equivalency of structures. It is possible, for example, that pH_i modulates these channels through entirely different mechanisms. Consistent with this, replacement of K₈₀ with either of the fish residues does not support pH gating of mammalian ROMK (114). Structural models place K₈₀ in TM1, where it clearly is apart from the gating mechanism (see above). Given all the evidence to date, we should be open to the possibility that it also acts as the key pH sensor. Hydrogen bonding with A₁₇₇ and electrostatic interaction (Fig. 5) with the lipid bilayer may be sufficient to explain how K₈₀ is titrated at neutral pH. Obviously, further studies are required to determine whether K₈₀ acts as both the pH sensor and the pH gate.

PKA PHOSPHORYLATION SITES.

The two key PKA phosphorylation sites that are responsible for controlling channel P_o are strategically located in the cytoplasmic domain (Fig. 7). S₂₁₉ is situated at the apex of the domain between a PIP₂ binding residue, the G loop, and an intersubunit (R₃₁₁-E₃₀₂) salt bridge. S₃₁₃ is centrally positioned on the surface of the domain, where it is sandwiched between the intrasubunit (R₄₁-E₃₁₈) and intersubunit (R₃₁₁-E₃₀₂) salt bridges. The juxtaposition of the phosphorylatable serines with the PIP₂ and pH gating determinants provides a structural explanation for the function coupling between PKA phosphorylation and PIP₂-channel interaction (75) and pH-dependent gating (69). Importantly, several Bartter's disease-causing mutations have been identified that remove the phosphorylatable residues [S₂₁₉R (128) and S₃₁₃C (131)], alter nearby residues [L₂₂₀F (140)], or disrupt the intersubunit salt bridge R₃₁₁W (107, 123), and thereby alter the regulated gating process.

Fig. 7.

Location of PKA phosphorylation sites S₂₁₉ and S₃₁₃ (red) and intrasubunit (R₄₁-E₃₁₈) and intersubunit salt bridges (R₃₁₁-E₃₀₂). Cytoplasmic domains of 3 subunits are shown. Side chains of relevant residues are highlighted. Alphabetical labeling of residues reflects subunit designations (a, b, or c). K₂₁₈ (green) is a PIP₂ binding residue. G loops are shown in magenta.

Coupling Ligand Binding and Phosphorylation to Gating

Just how ligand binding and posttranslational modification of the cytoplasmic domain is transmitted to the movement of the transmembrane helix remains a central unresolved question in the K_ir field. Several mechanisms have been suggested. In ROMK, conformational changes in the cytoplasmic domain are associated with gating (124). The G loop and points of inter- and intrasubunit interaction (31, 32, 71, 114) have been implicated in coupling these changes to the transmembrane pore. Furthermore, because electrostatic interactions between potassium and the channel may occur over long distances, subtle conformational changes at a single site in the long cytoplasmic potassium conduction pathway can potentially affect potassium transport energetics over the entire pore (117).

Summary

The molecular mechanisms that ultimately maintain sodium and potassium homeostasis reside in the tubular epithelial cells of the kidney. Since the initial discovery of the ROMK channel, it has become clear that the predominant ionic pathways necessary for luminal K⁺ recycling and secretion, as well as for K⁺ movement across basolateral membranes, take the form of highly regulated K⁺-selective channels that belong to the inward-rectifier K⁺ channel family. ROMK represents the prototypic member. ROMK isoforms are differentially expressed along the TAL and distal nephron, where they form apical, weak inward-rectifier K⁺ channels. In TAL cells ROMK channels mediate the K⁺ efflux required by NKCC2 for NaCl transport while contributing to paracellular Na⁺ and Ca²⁺ reabsorption. Loss-of-function mutations result in type II Bartter's syndrome, confirming the dependence of NaCl reabsorption on ROMK-mediated K⁺ efflux. More distally, these channels establish an exquisitely regulated K⁺ secretory pathway essential for K⁺ homeostasis.

ROMK channels, similar to their inward-rectifier cousins, are extraordinarily complex molecular machines whose constituent subunits undergo complex cooperative and dramatic conformational changes, exhibit modulatory intersubunit interactions, and interact distinctively with numerous regulatory factors. A multitude of intracellular factors and processes including Mg²⁺, polyamines, protons, ATP, PIP₂, cAMP-dependent protein kinase, protein kinase C, PTK, SGK-1, and WNK kinases (WNK4, L-WNK1) participate in the remarkably intricate regulation of these channels. The complexity of the ATP sensitivity of these channels is illustrated by their interactions with CFTR through scaffolding proteins and hence divergence from classical ATP-sensitive potassium channels. Conceptually more far-reaching has been the structure of the ROMK channel protein as a simple two-transmembrane unit flanking a “pore loop.” With four such subunits surrounding a highly conserved ion conduction pathway consisting of opposing P loops, ROMK, as well as K_ir2.1 and K_ir3.1, yielded at the time of their discovery the very first glimpse into nature's most basic and fundamental molecular blueprint for a self-contained potassium-selective pore. Such an architectural plan is found embedded and reproduced in all six-transmembrane, two-transmembrane, and four-transmembrane two-pore potassium channels.

GRANTS

This review was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-54231, DK-63049) and the American Heart Association (GIA0855321E).