The Molecular Biology of Renal K + Channels

Renal epithelial transport depends on the coordinated function of potassium channels with ion transporters (co-transporters, channels, and exchangers) and ion pumps in apical and basolateral membranes of distinct cell types along the nephron of the mammalian kidney. Potassium (K ⁺ ) channels are key members of this integrated transport system in renal epithelial cells. First, renal K ⁺ channels participate in generating cell membrane potential; since numerous transporters are electrogenic, changes in cell membrane potential could alter the transport rate of a given substance. Second, renal K ⁺ channels are involved in the volume regulation that is essential for preventing cell swelling or shrinking in the hypotonic or hypertonic environment. Third, renal K ⁺ channels play an important role in K ⁺ recycling which is essential for maintaining the function of several transport proteins, such as Na ⁺ -K ⁺ -ATPase. Finally, renal K ⁺ channels are extremely involved in K ⁺ secretion in the aldosterone-sensitive distal nephron (ASDN). Figure 47.1 is a scheme providing an overview regarding the role of K channels in different renal segments.

Introduction

Figure 47.1, A cell model demonstrating the role of K channels in different renal segments.

Since K ⁺ channels play such an important role in kidney function, understanding the structure and regulation of renal K ⁺ channels is essential to gaining insights into the molecular mechanisms of kidney potassium handling. In the past decades, the development of molecular biology and patch-clamp techniques has had a significant impact on the exploration of the molecular identity of some renal K ⁺ channels. This chapter summarizes our current understanding of the molecular identity of renal K ⁺ channels, and will specifically focus on the ROMK (Kir1; KCNJ1 ) channel. We will discuss similarities, as well as certain differences, in the properties of cloned K ⁺ channels compared to native K ⁺ channels expressed in the different nephron segments.

The Molecular Biology of ROMK, a Distal K ⁺ Secretory Channel

Structure of Inward Rectifying K ⁺ Channels and ROMK

The K ⁺ channel, ROMK (Kir1; KCNJ1 ), belongs to a growing family of inwardly rectifying K ⁺ (Kir) channels that are functionally characterized by high potassium selectivity and by either weak or strong inward rectification. To date, 16 Kir genes have been identified and classified into seven subfamilies (Kir1.x to Kir7.x). All Kir channels have a membrane topology consisting of two membrane-spanning domains (M1 and M2), an intervening H5 pore-forming region, and cytoplasmic amino (NH ₂ -) and carboxyl (COOH-)-terminal domains ( Figure 47.2 ). This membrane topology corresponds to the last two membrane spanning segments of the voltage-gated K ⁺ channels, suggesting a common ancestral origin. The N- and C-terminal cytoplasmic regions of Kir channels provide regulatory domains ( Figure 47.2 ) that can be phosphorylated by kinases, and that interact with protons, nucleotides, and phosphoinositides.

Figure 47.2, Topology of ROMK (Kir1.1) K + channel.

The three-dimensional structure of ROMK is not available, but it can be inferred from X-ray crystallographic structures of bacterial and eukaryotic K ⁺ channels. The first crystal structure of K ⁺ channel determined is from Streptomyces lividans (KcsA). Although not an inward rectifier K ⁺ channel, KcsA shares the same membrane topology with Kir channels with two membrane-spanning M1 and M2 segments, a H5 pore-forming region, and N- and C-terminal cytoplasmic domains. The amino acid sequence of KcsA is similar to the corresponding region of other K ⁺ channels, including vertebrate and invertebrate voltage-gated K ⁺ channel, inward rectifier, and calcium-activated K ⁺ channels. The crystal structure of KcsA reveals a tetramer with four identical subunits that encircle a central ion conduction pathway with four-fold symmetry ( Figure 47.3 ). The M1 and M2 segments of each subunit form α-helices and are inserted into the tetramer such that M2 faces the central ion conduction pathway (termed inner helix), and M1 faces the lipid membrane (outer helix). Amino acids connecting M1 and M2 (from N to C) form the turret, a tilted pore helix that runs half-way through the membrane, and the selectivity filter bearing the Thr-X-Gly-Tyr-Gly (X is any amino acid) signature sequence of all K ⁺ channels. The inner helices are tilted with respect to the membrane, so that the subunits open like the petals of a flower facing the outside of the cell. Furthermore, the four inner helices pack against each other as a bundle near the intracellular aspect of the membrane, giving the appearance of an inverted teepee.

Figure 47.3, Architecture of inwardly rectifying K (Kir) channels 276.

The ion conduction pathway (pore) of KcsA can be functionally divided into three parts that (from the outside of the cell) consist of the selectivity filter, a water-filled wide cavity near the middle of the membrane (the central cavity; ~10Å across), and the internal part of the pore made up of the anti-lipid facing amino acids of four inner helices. The selectivity filter (~12Å in length) is the narrowest part of the pore, and is lined by four evenly spaced layers of carbonyl oxygen atoms from amino acids X-Gly-Tyr-Gly, and a single layer of hydroxyl oxygen atoms from amino acid threonine. K ⁺ ions dehydrate and enter the selectivity filter in single file. The five layers of oxygen atoms form four consecutive K ⁺ ion-binding sites to stabilize dehydrated K ⁺ ions in the filter, which compensates for the energy required for dehydration. Due to repulsion between closely spaced ions, K ⁺ ions occupy only two of the four binding sites at a given time, in either 1,3 or 2,4 configuration. These unique structural and energetic features contribute to the extremely high selectivity (~1000 to 1 for K ⁺ over Na ⁺ ) yet fast conduction rate (up to ~10 ⁸ ions/sec) of K ⁺ channels for K ⁺ ions.

The crossing of four inner helices of KcsA as a bundle near the intracellular aspect of the membrane creates a point of structural constraint between the membrane pore and the cytoplasm, referred to as bundle crossing. Structural comparison of KcsA with Mthk, a bacterial calcium-activated K ⁺ channel, provides insights into how bundle crossing might function as an activation gate for K ⁺ channels. In KcsA, the inner helices are straight and the diameter of bundle crossing is about 3.5Å, suggesting that the crystal structure of KcsA is in closed conformation. In contrast, the crystal structure of Mthk, which is solved in the presence of high (Ca ²⁺ ), thus likely in the open conformation, reveals that inner helices are bent at a hinge point and splayed open so that the bundle crossing does not form a barrier to the flow K ⁺ ions between the central cavity and the cytoplasm. Thus, KcsA and Mthk structures likely represent closed and open pore conformations of many different K ⁺ channels, including ROMK. Conservation of a glycine residue at the inner helix hinge point in most K ⁺ channels supports this conclusion.

The structural studies of KcsA and Mthk do not include the cytoplasmic N- and C-terminal domains. The crystallographic structure of the bacterial inward rectifier K ⁺ channel KirBac1.1, including the transmembrane and cytoplasmic domains, provides the first insight into the intracellular domains of Kir channels. The structures of the intracellular domains of eukaryotic Kir channels are determined in two ways. One uses only the N- and C-terminal cytoplasmic domains connected by an artificial kinker for crystallographic studies. The other determines the structures of cytoplasmic domains, together with the transmembrane domains, using either a chimeric channel containing the outer three quarters of the membrane domains of KirBac3.1 and the inner one quarter of the membrane domain and the intracellular N- and C-terminal domains of Kir3.1 or the native form of inward rectifier K ⁺ channel from chicken (Kir2.2). Overall, these studies reveal that the N-terminal cytoplasmic domain of one subunit interacts with the C-terminus of the adjacent subunit, and together form predominantly β-sheet structures. The four sets of associated N- and C-terminal domains assemble into a tetramer surrounding a water-filled cytoplasmic pore that extends coaxially from the transmembrane pore domain. Thus, the cytoplasmic pore extends the ion permeation pathway to ~60Å, nearly twice the length of the transmembrane pore. The apex of the cytoplasmic pore abutting the transmembrane domain is formed by a loop referred to as the “G-loop.” In the crystal structures, the G-loops are in either a constricted or dilated conformation, likely representing closed and open states of the cytoplasmic pore, respectively. Thus, Kir channels contain two regions along the length of ion conduction pathway that can adopt constricted or dilated conformation and function as the gates. One is the inner helix bundle crossing, and the other is the apex of the cytoplasmic pore formed by the G-loops. Interestingly, the plasma-membrane facing surface of the cytoplasmic pore contains many positively charged amino acids known to be important for binding to membrane phosphatidylinositol-4,5-bisphosphate (PIP ₂ ) (see section on “Regulation of ROMK by PIP ₂ “ below). Interaction between these amino acids and membrane PIP ₂ may alter the conformation of the G-loop and the inner helix bundle crossing to activate the channel.

ROMK Channel Isoforms

Following the cloning of ROMK1 (Kir1.1a) from rat kidney, three additional alternatively spliced forms of this channel were isolated ( Figures 47.2 and 47.4 ), and named ROMK2 (Kir1.1b), ROMK3 (Kir1.1c) and ROMK6 (Kir1.1d). The encoded ROMK proteins differ at the beginning of the N-terminus – ROMK2 (also rat ROMK6, which has the same amino acid sequence as ROMK2; Figure 47.4 ) has the shortest N-terminus, and splicing adds either 19 or 26 amino acids for ROMK1 or ROMK3, respectively (see Figures 47.2 and 47.4 ). Relative ROMK mRNA abundance measured by competitive PCR has shown that ROMK2 and ROMK3 are much more abundant than ROMK1 or ROMK6 in rat kidney. In addition, a novel set of ROMK proteins, about one-third the size of native ROMK, has been suggested to be formed from alternative splicing of the ROMK core exon. The significance of these putative smaller channel proteins remains unclear. Six splice variants have been identified in the human ROMK gene, KCNJ1 , located on chromosome 11q24. These six human transcripts apparently encode only three distinct polypeptides, two of which are similar to rat ROMK1 and ROMK2. A rat homolog of the third human ROMK isoform has not been identified. Two ROMK homologs have also been cloned from human kidney, but their roles in renal function are unknown.

ROMK Channel Localization

Rat ROMK1-3 are differentially expressed along the nephron from the thick ascending limb of Henle, TAL, to the outer medullary collecting duct, OMCD, ( Figure 47.5 ). The rat TAL and distal convoluted tubule, DCT, express ROMK2 and ROMK3 messenger RNA, while principal cells in the cortical collecting duct, CCD, express ROMK1 and ROMK2 transcripts (see Figure 47.5 ). The outer medullary collecting duct cells appear to express only ROMK1 transcripts. The general single channel properties of the ROMK1, 2, and 3 isoforms are similar, e.g., single channel conductance and open probability ( Figure 47.4 ). Although the specific functional/regulatory consequences of the different isoforms have not been fully-elucidated, a serine at the fourth position in the extended N-terminus in ROMK1 has been shown to be required for sensitivity to arachidonic acid ( Figures 47.2 and 47.4 ) and protein kinase C (see section on ROMK function below). Thus, ROMK1 may add distinct functional characteristics to ROMK channels. No specific role for the extended N-terminus of ROMK3 has yet been identified. Whether tetrameric ROMK channels are formed of different subunits (e.g., heterotetramers of ROMK2 and ROMK1 in the cortical collecting duct) or exist only as homotetramers is not known. Finally, ROMK transcripts are present in some other tissues, including the early gravid uterus. Roles for ROMK in these tissues have not been determined.

Figure 47.5, The distribution of the ROMK 1, 2, and 3 isoforms along the rat nephron.

Antibody generated to sequences of ROMK shared by all isoforms has demonstrated an apical pattern of channel protein expression in rat TAL (including macula densa cells), DCT, and early CNT cells, and principal cells of the CCD and OMCD. This localization is consistent with the ROMK channel providing a K ⁺ secretory pathway in these renal epithelia.

ROMK Channel Properties are Similar to the Distal (SK) K ⁺ Secretory Channel

The single channel characteristics and regulatory properties of ROMK channels expressed in X. laevis frog oocytes are virtually identical to those of the native ATP-sensitive, small conductance (SK) channels in TAL cells ( Figure 47.6 ) and principal cells in the CCD ( Figure 47.7 ). Similar kinetic characteristics of K ⁺ , NH ₄ ⁺ , and Tl ⁺ have been observed in the native secretory K ⁺ channel in the rat CCD and ROMK2 channels expressed in X. laevis oocytes, leading Palmer and co-workers to conclude that the native SK and cloned ROMK channels were identical. A further characteristic of the low conductance secretory K ⁺ channel found in principal cells is a lack of sensitivity to external TEA ⁺ .

Figure 47.6, Model for ion transport in the thick ascending limb.

Figure 47.7, Model for ion transport by the principal cell in the collecting duct.

The general properties of ROMK channels expressed in Xenopus oocytes include: (1) weak inward rectification ( Figure 47.4 ) that is dependent on the binding of cytosolic Mg ²⁺ or other polyvalent cations to the channel pore ; (2) activation by protein kinase A-dependent phosphorylation processes ; (3) inhibition by high concentrations of MgATP ; (4) inhibition by slight reductions in cytosolic pH ; and (5) inhibition by arachidonic acid and protein kinase C. When coupled with gene expression and protein localization studies, these functional similarities strongly suggest that ROMK makes up the pore-forming subunit of the renal distal SK potassium channel.

Characteristics of the ROMK Channel Pore

Channel Kinetics

ROMK channels are characterized by a high open probability (P _o ) of greater than 0.9 for inward K ⁺ flux. The high open probability results from one open state and two closed states. One closed state is very short (~1 ms; 99% frequency), and the other is longer (~40 ms) but very infrequent (~1%). The infrequent closed state is due to blocking by divalent cations, as it can be abolished by EDTA. Choe and co-workers have also suggested that the closed state of ROMK results from K ⁺ ions transiently blocking its own pathway. Such a model would not require large molecular motions, but rather small molecular oscillations.

Channel Rectification

One of the fundamental characteristics of ROMK, as well as all Kir channels is inward rectification, the property of passing current more easily in the inward than in the outward direction (see Figure 47.4 ). Although this seems to be contrary to the role of ROMK in K ⁺ secretion, the inward rectification observed with ROMK, and with the kidney K ⁺ secretory channel, is “weak.” The term “weak” rectification refers to the ability of ROMK to actually pass outward current, albeit to a lesser extent than inward current. Many of the other Kir channels are “strong” rectifiers, and characteristically pass little outward current. The very high open probability of the ROMK channel, usually >0.9, may help make up for the rectification effect on K ⁺ secretion. In other words, although the outward conductance (ease of passing K ⁺ secretory current) is less than the inward conductance, the channels are open most of the time, and thus are able to secrete large amounts of K ⁺ . We now know that inward rectification of ROMK is due to blocking of the channel pore by Mg ²⁺ or polyamines like spermine or spermidine from the intracellular side. Thus, it is possible that variations in the cytosolic concentrations of these inorganic and organic cations could provide an important mechanism regulating outward (i.e., K ⁺ secretory current. It was suggested that the inhibition of outward K ⁺ fluxes through ROMK by Mg ²⁺ may contribute to K ⁺ -wasting in the setting of Mg ²⁺ deficiency. A subsequent study by Yang et al. examining the regulation of ROMK expressed in Xenopus oocytes and the native channel by intracellular and extracellular Mg ²⁺ supports this idea.

Kinetic studies of inward rectification by Mg ²⁺ and polyamines indicate that the effect is voltage-dependent and depends on the extracellular concentration of K ⁺ (thus varies with the K ⁺ reversal potential in the constant intracellular (K ⁺ )). These findings suggest an interaction between permeant and blocking ions, and the presence of a variable energy well within the channel pore. The crystal structure of Kir revealing a long channel pore with multiple binding sites for K ⁺ and blocking ions is consistent with this idea.

Electrophysiological studies have demonstrated the importance of the M2 segment and COOH-termini in determining the inward rectifying characteristics of inwardly rectifying K ⁺ channels. Two residues are particularly important in determining whether the rectification is strong or weak. In strong inward rectifiers like IRK1 (Kir2.1), a negatively charged residue, aspartic acid (D172 in IRK1), in the M2 membrane segment has been shown to be critical for strong inward rectification. In ROMK the aspartate residue is replaced by asparagine (see Figure 47.8 , IR Site #1, N171 in ROMK1), consistent with the weak rectification of this channel. A second residue located in the C-terminus has also been shown to be an important contributor to strong rectification in IRK1. This glutamate residue (E224 in IRK1) is replaced by a glycine residue in ROMK (see Figure 47.8 ; IR Site #2). This C-terminal glycine residue in ROMK is a part of the Walker A site that contributes to the nucleotide-binding interactions (see Figure 47.1 , and IR Site #2 in Figure 47.8 ) in the nucleotide-binding domain (NBD; Figure 47.8 ), and thus serves a different gating function in ROMK. As expected from this model, exchange of the ROMK C-terminus with that on IRK1 produces strong rectification in oocytes injected with the mutant ROMK channel. Consistent with the electrophysiological results, the crystal structure of Kir2.2 reveals that the inner helices line the central cavity and the internal half of the membrane pore, and the side chain of D172 points to the center of the central cavity. The binding sites for Mg ²⁺ in the crystal structure of Kir2.2 were examined by soaking the crystal with 10 mM Sr ²⁺ , an electron dense mimic of Mg ²⁺ . Three density peaks due to Sr ²⁺ are observed in the crystal of Kir2.2: one in the central cavity corresponding to the position of D172 and two in the cytoplasmic pore, referred to as the upper ring and lower ring of charges, respectively. The upper ring of charges consists of E224 of IRK1 and an additional glutamate residue E229 from each of four subunits. The lower ring of charges consists of four D255, one from each subunit.

Figure 47.8, The major functional and regulatory sites, and the Bartter’s mutations are shown in this schematic representation of ROMK1, Kir1.1a

Finally, two different extracts of venom have been suggested to specifically inhibit ROMK channels. Both the snake toxin, δ-dendrotoxin, the honey bee venom extract, tertiapin, and the modified compond, tertiapin-Q appear to block ROMK activity by interacting with channel pore. Tertiapin-Q specifically blocks ROMK and Kir3.1, but not other Kir channels such as Kir4 and Kir5, and has been explored to estimate the density of ROMK channels in the CCD and CNT in response to dietary K ⁺ intake. A study, however, indicates that tertiapin-Q can also block MaxiK (or BK, hSlo1 ) Ca ²⁺ -activated K ⁺ channels. Thus, caution should be exercised when utilizing tertiapin-Q to define ROMK activity under the condition in which MaxiK channels are activated by flow.

Regulation of the ROMK K ⁺ Channel

ROMK channel activity, like that of the native SK channel in TAL and principal cells, is regulated by a variety of factors that either activate or inhibit channel activity ( Figure 47.9 ). The molecular mechanisms for these alterations in channel function are rapidly being identified.

Figure 47.9, The major identified regulators of ROMK channels.

Protein Kinase A (PKA)

PKA-dependent phosphorylation processes activated by receptor-mediated events or alterations in cytosolic second messengers (e.g., cyclic AMP, cAMP) play important roles in regulating the native SK channel in principal cells of the CCD ( Figure 47.10 ). Phosphorylation-dephosphorylation processes also modulate the activity of the cloned ROMK K ⁺ channel. K ⁺ channel activity in excised inside-out patches of oocytes expressing ROMK requires activation by PKA-dependent phosphorylation processes. Rundown or loss of ROMK channel activity in these patches occurs whenever phosphatase-mediated dephosphorylation activity is greater than phosphorylation (see also section on PIP ₂ below for role of protein versus lipid kinases and phosphatases in the process of rundown and reactivation by MgATP).

Figure 47.10, A cell model illustrating the mechanism by which ROMK channel activity is regulated by the interaction among c-Src, SGK1, and WNKs in the CCD.

The critical PKA phosphorylation sites are on the channel protein itself (see Figure 47.2 ). This has been demonstrated by several observations. First, ROMK protein expressed in HEK-293 cells can be phosphorylated by PKA. Phosphopeptide analysis and mapping have shown three serine residues phosphorylated by PKA (one residue on the N-terminus (serine 25 in ROMK2) and two residues on the C-terminus (serine 200 and serine 294 in ROMK2)). Mutation of any single PKA phosphorylation site on ROMK2 reduces whole cell K ⁺ currents by 35–40% in oocytes; mutation of two or more of the three sites produces non-functional channels. This is consistent with the critical role of PKA phosphorylation in channel activation. Second, at the single channel level the N-terminal and C-terminal PKA phosphorylation sites alter the channel activity, albeit differently. None of the mutations with serine residues replaced by alanine alters the single channel conductance. Each of the C-terminal PKA phosphorylation site mutations, however, reduces open probability (P _o ) by about 40%, due to the appearance of a new long closed state. This reduction in P _o is sufficient to account for the observed reduction in whole oocyte currents. Replacing the N-terminal serine with alanine does not change P _o , but does reduce the probability of finding functioning channels by about 60%. The mechanism for this reduction in active channels is not known at present. The mechanism by which PKA increases ROMK channel activity may include stimulation of surface expression and enhance the effect of PIP ₂ on ROMK channels. It has been shown that stimulation of PKA increases the sensitivity of ROMK channels to PIP ₂ in Xenopus oocytes. Also, a recent study demonstrates that mutation of serine residue 44 to aspartate increases the surface delivery of ROMK1 channels. One of the fundamental characteristics of SK channels in principal cells and in the TAL is their activation by Gs-coupled receptors or by the addition of cyclic AMP. AKAPs are A - K inase A nchoring P roteins that bind PKA holoenzyme (catalytic plus regulatory subunits), and maintain the enzyme at specific intracellular sites. Wang and co-workers reported that ROMK1 channels expressed in X. laevis oocytes could not be activated by cyclic AMP unless expressed with an isoform of AKAP, AKAP79. On the other hand, findings that ROMK channels carrying PKA site mutations exhibit reduced current in Xenopus oocytes in the absence of AKAP79 suggests that ROMK can be phosphorylated without AKAP79. The role of AKAP in the native tissue is not determined. Some studies suggested that the Na/H exchange regulatory protein 2 (NHERF-2) might act as an AKAP, because NHERF-2 interacts with ROMK channels and facilitates the stimulatory effect of SGK1 on ROMK channels.

Arachidonic Acid (Aa)

Like the native SK channel in the CCD, ROMK1 channels expressed in Xenopus oocytes are sensitive to arachidonic acid (AA ). The effect of AA is specific, since other fatty acids failed to mimic the effect of AA. However, AA has little-to-no effect on the other two ROMK family members, ROMK2 and ROMK3. Since the amino acid sequences of the ROMK channels are identical, with the exception of the N-terminus, the role of the N-terminus in mediating the effect of AA is strongly suggested. This is supported by the demonstration that deletion of the initial 37 aa of ROMK1 abolished the effect of AA. Moreover, a serine residue at the fourth position within the N-terminus of ROMK1 has been shown to play a crucial role in the AA-mediated inhibition of ROMK1, since mutation of this serine residue to alanine abolished the effect of AA ( Figures 47.1 and 47.3 ). Since this serine residue is a putative PKC phosphorylation site, and AA has been shown to activate PKC, the effect of AA may depend on stimulation of a membrane-bound PKC.

Protein Kinase C (PKC)

Activation of PKC phosphorylation processes inhibits the apical SK channel in the CCD. ROMK1, which is exclusively expressed in collecting ducts, has three potential PKC phosphorylation sites involving serine residues: one on the N-terminus; and two on the C-terminal end. ROMK2 and ROMK3 only have the two C-terminal PKC phosphorylation sites (see Figures 47.2 and 47.4 ). Using an in vitro phosphorylation assay, it was observed that serine residue 4 in the N-terminus and serine residue 201 in the C-terminus are two major PKC-induced phosphorylation sites. The effect of PKC on ROMK channels is complex. It was demonstrated that phosphorylation of either serine residue 4 or 201 is essential for ROMK1 export to the plasma membrane. On the other hand, stimulation of PKC in vivo has been shown to inhibit ROMK channel activity. The N-terminal serine residue at the fourth position appears to be most important to PKC-mediated K ⁺ channel inhibition of ROMK1. Interestingly, this is the same residue critical for the inhibitory effect of arachidonic acid (see Figure 47.4 ). However, it is possible that PKC-induced inhibition of ROMK channels may be indirect, resulting from a decrease in PIP ₂ content. It was demonstrated that stimulation of PKC decreases the PIP ₂ level in the plasma membrane. Because PIP ₂ is essential for maintaining ROMK channels in the open state, decreases in PIP ₂ levels may contribute to the PKC-induced inhibition.

WNKs (with No Lysine Kinase)

WNKs are a family of four serine/threonine protein kinases named WNK1–4. Among them, WNK1, 3, and 4 are expressed in the distal nephron. The discovery that mutations in WNK1 and WNK4 cause the autosomal-dominant hypertension and hyperkalemia known as pseudohypoaldosteronism type 2 (PHA2) led to extensive characterization of their properties and function. Evidence indicates that the WNK family plays an important role in the regulation of ROMK channels. WNK1, 3, and 4 inhibits the ROMK channel activity ( Figure 47.10 ), and the effect of WNKs on ROMK is mediated by stimulation of clathrin-dependent endocytosis. Intersectin, a scaffold protein containing two Eps15 homology domains and four or five tandem SH3 domains, interacts with WNK1 and 4, which is required for the regulation of clathrin-mediated endocytosis of ROMK by WNKs. In addition, a clathrin adaptor protein, Autosomal Recessive Hypercholesterolemia (ARH), has been shown to interact with ROMK, and the interaction may be involved in the stimulation of endocytosis of ROMK by WNK1. Dysregulation of ROMK by WNK1 and 4 may contribute to the hyperkalemia in PHA2.

A kidney-specific splice form of WNK1 (KS-WNK1), in which an alternative 5′ exon replaces the first four exons of WNK1, is expressed in the distal nephron. Unlike the long form of WNK1, KS-WNK1 lacks kinase activity and, by itself, does not regulate ROMK channels. However, KS-WNK1 antagonizes the inhibitory effect of WNK1 on ROMK. It has been reported that high K ⁺ intake increases, whereas low K ⁺ intake decreases, the expression of KS-WNK1, and that increased KS-WNK1 expression attenuates the inhibitory effect of WNK1 on ROMK channels. Thus, the alteration of the ratio between the long and short form of WNK1 may be an important mechanism by which a dietary K ⁺ intake regulates ROMK channel activity.

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Introduction

The Molecular Biology of ROMK, a Distal K + Secretory Channel

Structure of Inward Rectifying K + Channels and ROMK