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Molecular Diversity and Regulation of Renal Potassium Channels

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, and Department of Medicine, Yale University and VA CT Medical Center, West Haven, Connecticut; and Department of Pharmacology, New York Medical College, Valhalla, New York

ABSTRACT

I. INTRODUCTION

A. Physiological Roles for K+ Channels in Kidney

1. Overview of renal K+ transport in K+ homeostasis

2. Other physiological roles for K+ channels in kidney

B. Overview of Renal K+ Channel Diversity and Structure

1. Evolution and diversity

2. Structural classification and functional domains

3. The pore region: an essential feature of K+ channels

4. K+ channels are heteromultimeric complexes

II. FUNCTION AND REGULATION OF POTASSIUM CHANNELS IN THE KIDNEY

A. Epithelial K+ Channels

1. K+ channels along the nephron

A) FUNCTION OF K+ CHANNELS IN PROXIMAL TUBULE.

B) FUNCTION OF K+ CHANNELS IN THE TAL.

C) FUNCTION OF K+ CHANNELS IN THE MAMMALIAN DISTAL CONVOLUTED TUBULE.

D) FUNCTION OF K+ CHANNELS IN THE COLLECTING DUCT.

2. K+ channels in cultured renal epithelial cells

A) PROXIMAL TUBULE CELL CULTURE.

B) DISTAL TUBULE CELL CULTURE.

B. Nonepithelial K+ Channels

1. Mesangial K+ channels

2. K+ channels in juxtaglomerular apparatus

3. Vascular K+ channels

C. Major Physiological Role of K+ Channels in Renal Epithelial Function

1. K+ secretion

A) EFFECT OF K+ DIET.

B) EFFECT OF ACID AND BASE BALANCE.

C) HUMORAL REGULATION.

D) PATTERN OF MATURATION.

2. Volume regulation

D. Pattern of Regulation of K+ Channels

1. Pattern of regulation of apical K+ channels in principal cells

2. Pattern of regulation of basolateral K+ channels

III. MOLECULAR PHYSIOLOGY OF ROMK CHANNELS

A. ROMK Channel Structure and Function

1. ROMK channel structure

2. ROMK channel isoforms

3. ROMK channel localization

4. Characteristics of the ROMK channel

A) CHANNEL KINETICS.

B) CHANNEL RECTIFICATION.

C) INHIBITORS OF ROMK.

B. Regulation of the ROMK Channel Is Similar to the Distal Small-Conductance (35-pS) K+ Secretory Channel

1. Protein kinases and phosphatases

A) PKA.

B) SGK.

C) PKC.

D) WNK.

E) PTK-PTP.

2. Lipids and products of lipid metabolism

A) ARACHIDONIC ACID.

B) PHOSPHATIDYLINOSITOL PHOSPHATES (PIP2).

3. Cytosolic pH

4. Nucleotide sensitivity and CFTR

A) EVIDENCE THAT AN ABC PROTEIN FORMS A SUBUNIT OF THE 35-PS K+ CHANNEL.

B) FUNCTIONAL CONSEQUENCES OF ABC PROTEIN-ROMK INTERACTIONS.

C) CFTR, ROMK, AND THE EPITHELIAL SODIUM CHANNEL INTERACTIONS.

C. Regulation and Developmental Changes of ROMK Expression

1. Regulation

2. Developmental expression of ROMK

D. Lessons From Bartter's Syndrome

1. Bartter's syndrome: a TAL tubulopathy

2. The mouse ROMK knockout

IV. OTHER POTASSIUM CHANNELS IN KIDNEY

A. 6-TM Renal K+ Channels

1. Proximal tubule

2. Distal tubule

B. 2-TM Renal K+ Channel (Non-ROMK)

1. Kir2.3 channel

2. Kir4.1/4.2 and Kir5.1 channels

3. Kir6.1 channel

4. Kir7.1 channel

C. 2-Pore K+ Channels (K2P)

D. 1-TM (CHIF, Channel Inducing Factor)

V. CONCLUSIONS AND FUTURE DIRECTIONS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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A. Physiological Roles for K⁺ Channels in Kidney

1. Overview of renal K⁺ transport in K⁺ homeostasis

The physiological regulation of the body's internal and external K⁺ balance depends on maintenance of unequal distribution between the intra- and extracellular fluid compartments and on effective renal excretion. Constancy of the small extracellular pool of K⁺ and its low extracellular concentration depend ultimately on effective mechanisms of regulated renal K⁺ excretion. Renal K⁺ excretion is the result of three mechanisms: free filtration in the glomerulus, extensive reabsorption in the proximal nephron segments, and controlled net K⁺ secretion in distal nephron largely determining K⁺ excretion (164, 165, 169, 593). K⁺ reabsorption in certain distal nephron segments has also been observed in states of K⁺ deficiency.

Figure 1 provides an overview of K⁺ transport along the nephron. The bulk of filtered K⁺ is reabsorbed as tubule fluid passes through the proximal convoluted tubule. Some K⁺ enters the descending segments of the loop of Henle passively because of the increasing K⁺ concentration along the corticomedullary axis (15). K⁺ leaves the ascending limb of Henle, again passively, as K⁺ declines in the interstitium towards the corticomedullary region. K⁺ is further reabsorbed in the thick ascending limb of Henle (TAL) by a specific cotransporter (Na⁺-K⁺-2Cl^–). Modest K⁺ secretion takes place in the distal convoluted tubule and is followed by regulated, more extensive secretion in the initial and cortical collecting ducts. The connecting tubule of deep juxtamedullary nephrons has also been shown to secrete K⁺ (227). The direction of K⁺ transport reverses as fluid passes the outer medullary and terminal papillary collecting duct. Variable net reabsorption has been demonstrated in these nephron segments (111). K⁺ reabsorption in the terminal segments of the nephron, together with passive entry of K⁺ into the descending limb of Henle, constitutes the pathways of K⁺ recycling in the renal medulla (15).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Potassium transport along a simplified nephron. K+ is filtered and extensively reabsorbed along the proximal convoluted tubule (PCT). Some K+ enters the proximal straight tubule (PST) and the thin, descending limb of Henle's loop (DTL). But extensive K+ reabsorption along the ascending thin limb of Henle (ATL) and along both the medullary (MTAL) and cortical (CTAL) portions of the thick ascending limb sharply reduce the amount of K+ by the time fluid has reached the beginning of the distal convoluted tubule (DCT). Regulated K+ secretion takes place in the connecting tubule (CNT), DCT, initial collecting duct (ICD), and cortical collecting duct (CCD), and reabsorption may occur in the terminal nephron segments, the outer (OMCD) and inner (IMCD) medullary collecting duct. The reabsorptive and secretory components of K+ transport in the loop of Henle and the further distally located nephron segments are subject to regulation, but transport in the proximal tubule does not control urinary K+ excretion. The arrow size represents relative magnitudes of K+ secretion and reabsorption.

Figure 2 shows the basic functions of K⁺ channels in various tubule cells along the nephron in the mammalian kidney. In addition to the roles of K⁺ channels in mediating net K⁺ secretion or absorption, these channels serve to 1) maintain the cell-negative potential at rest providing a driving force for electrogenic transport processes and stabilizing the membrane potential during large fluxes of cations into, or anions out of, tubule cells; 2) help in recovery of cell volume after swelling; 3) recycle K⁺ back to tubule fluid in the TAL where K⁺ entered cells via the Na⁺-K⁺-2Cl^– cotransporter; and 4) recycle K⁺ across basolateral membranes in conjunction with Na⁺-K⁺-ATPase turnover. Detailed descriptions of these functions for the various cells along the nephron are given in section II.

View larger version (33K): [in this window] [in a new window]   FIG. 2. A model of a single nephron showing the function of renal K+ channels in proximal tubules, thick ascending limb (TAL), and cortical and outer medullary collecting duct (CCD).

Although the molecular structure of many K⁺ channels has been defined, our knowledge of their location along the nephron and their polarized expression and functions in native renal epithelial cells is incomplete. The current state of our understanding of the distribution of specific K⁺ channel types along the nephron is shown in Figure 3. While the assignment of cloned K⁺ channels to specific functions in tubule cells remains a major problem, the exceptions are as follows. ROMK, an inwardly rectifying K⁺ channel with two membrane-spanning domains, has been identified as one of the main secretory K⁺ channels; Ca²⁺-activated maxi-K⁺ channels are involved in volume control and flow-dependent K⁺ secretion in the collecting duct; and voltage-sensitive K⁺ channels play a role in the stabilization of the membrane potential in the face of electrogenic transport processes.

View larger version (31K): [in this window] [in a new window]   FIG. 3. A model of a single nephron demonstrating the expression of currently known K+ channel genes.

K⁺ channels are transmembrane proteins that mediate passive K⁺ movement across cell membranes via a highly selective, aqueous pore. Molecular studies carried out over the past decade have firmly established that K⁺ channels constitute the largest and most diverse class of ion channels. These data are in agreement with numerous electrophysiological studies documenting the existence of a wide variety of K⁺ currents in virtually all cells examined to date. They are unique among cation channels since, unlike Na⁺ and Ca²⁺ channels, they are found in virtually all living organisms and are widely distributed among cells within each organism. It is likely, therefore, that other cation channels evolved from K⁺ channels and that ionic specificity for Ca²⁺ or Na⁺ was achieved by modifying the pore region (235).

In spite of an extensive knowledge of the cellular and tissue distribution of K⁺ currents and a rapidly growing understanding of the cellular physiology of K⁺ channels, biochemical and structural information lagged behind until 1987. The structure of the Na⁺ channel of excitable tissues had been determined more than 3 years previously (404, 406) using Na⁺ channel protein purified from the eel's electric organ, which expresses a large quantity of the protein. Conventional biochemical approaches failed to elucidate the structure of K⁺ channels, in large part because these proteins are expressed in very low amounts and are not readily amenable to large-scale purification, and high-affinity probes had not been yet developed.

A major breakthrough in understanding the molecular structure of K⁺ channels was achieved in 1987. Electrophysiological studies of Drosophila melanogaster mutants (Shaker) that exhibited leg shaking when exposed to ether, a volatile anesthetic, indicated the Shaker locus might encode a structural component of a voltage-dependent K⁺ (Kv) channel. A combination of mutation mapping and chromosome walking was used to successfully identify the Shaker gene (254, 441, 498, 551). Expression of the Shaker gene product in Xenopus oocytes confirmed it mediated Kv currents (553). Subsequently, several other Drosophila K⁺ channel genes (Shal, Shaw, and Shab) were isolated by homology cloning (71). These four proteins had a close evolutionary relationship as evidenced by a high degree of identity (40%) at the amino acid level in pairwise comparisons. The first mammalian K⁺ channel was isolated by homology cloning in 1988 and noted to be most closely related to Shaker (70% amino acid identity; Ref. 550). Subsequently, homologs of each Drosophila gene have been identified in mammals either by homology or expression cloning (190).

Although Shaker-related Kv channels were the first to be identified, they are by no means the only class of K⁺ channels and may not even be the most diverse group of K⁺ channels. Indeed, as will be discussed in more detail, three other major classes of K⁺ channels have been isolated, namely, the calcium-activated K⁺ channels (124, 274, 562), the inward rectifiers (213, 285), and the two-pore (2P) channels (174, 176, 309, 312, 415; see Fig. 4). The 2P channels appear to be the most diverse group of K⁺ channels with more than 50 members identified to date. Phylogenetic tree reconstruction, using deduced amino acid sequences of the various known classes of K⁺ channels, suggests that all known K⁺ channels arose from a common ancestor (101, 107, 237–239, 347). It is also noteworthy that the 2P channels are predicted to be more closely related to the small-conductance calcium-activated K⁺ channel (SK) family in spite of very different secondary structures: 2 pores and 4 transmembrane segments (TM) for TWIK and 1 pore and 6 TM for SK (101, 174, 312).

View larger version (41K): [in this window] [in a new window]   FIG. 4. Classification of K+ channels and schematic representation of their secondary structure. A: the -subunit of the 6-TM class of K+ channels: six transmembrane segments (S1-S6) and one pore (P). S4 represents the voltage sensor of Kv channels. The active channel is a tetramer and can also contain accessory () subunits. K+ channels belonging to this class include Kv1.1–1.10, IKCa, HERG, and KvLQT. B: unlike the other members of the 6-TM class, hSlo appears to have seven transmembrane segments (S0-S6) and the amino terminus is predicted to be located extracellulary. The S4 segment is well conserved, and the cytoplasmic tail contains a calcium-binding domain. The active channel is a heterotetramer and contains accessory () subunits. C: the -subunit of the 2-P class of K+ channels: 4 transmembrane segments (S1-S4) and 2 pores (P1, P2). These channels do not have an S4 segment. K+ channels belonging to this class include TWIK, TRAAK, TASK, and TREK. D: MinK and the MinK-related peptides (MiRPs) contain a single transmembrane segment and function as accessory subunits for a variety of 6-TM channels. E: the single transmembrane subunit MinK (KCNE1).

2. Structural classification and functional domains

Since a very large number a K⁺ channel genes have already been identified, deduced amino acid sequences and secondary structure information can be used to generate a structural classification. Three broad classes emerge from such analysis. The first consists of channels with six or seven transmembrane segments and one pore region (6-TM) including the Shaker-related Kv channels (190), KvLQT1 and Ca²⁺-activated K⁺ channels (KCa). A schematic representation of a prototypic 6-TM channel is shown in Figure 4A. The deduced amino acid sequences range from 450–900 amino acids in length (see Table 1). Hydropathy analysis indicates that they all contain six transmembrane segments. Because the proteins lack signal peptides, the amino and carboxy termini are both predicted to be located intracellularly. Figure 4B depicts the large-conductance voltage-gated K⁺ channel (hSlo), which unlike Kv has seven transmembrane segments and an extracellular amino terminus (138). Figure 4C shows the inward rectifiers (inward rectifier family; Kir1–7) that have two transmembrane segments and one pore (2-TM; Ref. 398). The amino and carboxy termini are both predicted to be located intracellularly and provide regulatory domains for modification by nucleotide, pH, phosphorylation, and phosphatidyinositol phosphates [e.g., phosphatidylinositol 4,5-bisphosphate (PIP₂)] and from heterotrimeric G protein-coupled receptors. This group includes ATP-sensitive channels and ATP-regulated channels such as ROMK (213). The most recently discovered group (Fig. 4D), and likely the most diverse subfamily, consists of K⁺ channels with four transmembrane segments and two pores (2-P; Refs. 309, 415). These channels appear to mediate background leak K⁺ currents in a variety of cells, and some currents are regulated by pH and membrane stretch. MinK or KCNE1 is single transmembrane subunit (Fig. 4E) that was the first "K⁺ channel" found in kidney but has subsequently been shown to function as a subunit of the Kv channel, KCNQ1.

All K⁺ channels mediate the rapid (>1 million ions/s) and selective transport of potassium. Before the cloning of the first K⁺ channel, a fairly comprehensive view of the pore and the selectivity filter was constructed based on electrophysiological data (203). It was envisioned that K⁺ entered the channel via a water-filled pore, wide on the outside but narrower as it reached the intracellular face of the membrane. The narrow passage facilitated interactions with stabilizing charges and conferred the high selectivity for K⁺. It was also predicted that the pore could accommodate several K⁺ lined in single file. These predictions proved to be remarkably accurate. Recent data, obtained from site-directed mutagenesis experiments of known K⁺ channels proteins (7, 8, 199, 263, 354, 355), have confirmed the model. Moreover, direct structural data not only validated the proposed structure but also provided an exquisitely detailed view of the pore (119, 189, 460). The 6-TM K⁺ channels contain a large hydrophobic core that thwarted repeated attempts at obtaining protein crystals suitable for high-resolution X-ray crystallographic studies. A bacterial channel, KCsA, of the 2-TM class was subsequently used to successfully generate protein crystals for high-resolution studies (119, 460). It is assumed that the model derived from these data is relevant to all known K⁺ channel pores. X-ray analysis of KCsA crystals revealed that K⁺ channels are tetramers. The four subunits interact to form a symmetrical structure at the center of which lies an aqueous pore. The critical structural elements of the pore consist of two membrane segments linked by a stretch of 25 amino acids, hydrophobic enough to partly penetrate the lipid bilayer, and also containing a highly conserved signature sequence: TxGYG. This latter sequence along with the last membrane segment (M2 and S6 for the 2-TM and 6-TM class) form the lining of the pore and determine permeation and selectivity. The pore is asymmetric and has a wide extracellular vestibule that narrows abruptly to 3 . This narrowing represents the selectivity filter and measures 15 in length. The pore then widens briefly, about halfway into the bilayer, to form a water-filled cavity 10 wide. The selectivity filter provides a binding pocket, lined by negatively charged backbone carbonyl groups of the signature sequence, which provides a better fit for a dehydrated K⁺ than for a Na⁺. The pore contains three of these K⁺ binding sites arranged in single file and in close enough proximity for electrostatic repulsion.

Crystallization of the KcsA pore under high and low K⁺ conditions indicates that the selectivity filter also functions as an outer gate for the channel (657). High K⁺ maintains the filter in an open configuration, and all four binding sites have similar electron density. Low K⁺ alters the structure of the filter and causes the two binding sites at opposite ends of the filter to have the highest electron density. K⁺ cannot move easily, and channel conductance is low. It has been proposed that K⁺ channels also possess an inner gate, formed by hinging of the inner transmembrane helix at a conserved glycine residue, and that sensors (such as voltage, nucleotides, calcium, and pH) may interact with the inner gate and regulate channel function (244). The KcsA pore and selectivity filter are similar to those recently shown for a bacterial Kir homolog Kirbac1.1 (296). This model also appears to provide a reasonable approximation of accurately the more complex pore/selectivity filter structures of the voltage-gated (Kv) 6-TM K⁺ channels based on the recent X-ray structure of a voltage-dependent K⁺ channel (KvAP) from Aeropyrum pernix (245). The selectivity filter of KvAP resembles that of KcsA. However, the structures diverge within the intracellular membrane leaflet, and in contrast to KcsA, KvAP has an opened pore. In addition, KvAP's pore is flanked by voltage "sensor paddles," which are postulated to move across the membrane and modulate the opening and closing of the pore.

Most K⁺ channels do not remain open indefinitely but alternate between open and closed states. The movement of K⁺ through these channels is governed by conformational changes resulting in channel opening or closing, also referred to as gating. K⁺ channels have evolved an array of gating sensors that respond to membrane voltage and/or a variety of extra- and intracellular molecules or ligands including protons, Ca²⁺, mono- and dinucleotides, cyclic nucleotides, and membrane phospholipids. For example, Kv channels have the ability to respond to membrane voltage. The fourth membrane domain is a critical part of a complex that participates in sensing changes in membrane voltage. The S4 segment stands out by virtue of having 6–9 positively charged amino acids (either lysine or arginine) out of 20. Membrane depolarization is believed to open the channel through an S4-mediated conformational change (256, 302, 318, 364, 425, 434, 514). The recent X-ray structure of the voltage-gated K⁺ channel has shown that S1-S4 helices are attached to the pore region and the latter half of S3 and S4 form the voltage sensor (245, 246).

It should be noted that there is at least one other mechanism for conferring voltage dependence. The 2-P channels, which lack an S4 segment, are thought to mediate background leak current. Recently, it was demonstrated that phosphorylation of a single amino acid of the 2-P channel, KCNK2 (expressed in the hippocampus), allows the channel to sense changes in membrane voltage (52). Furthermore, some 6-TM channels with conserved S4 segments lack voltage sensitivity (32, 46), consistent with voltage sensitivity depending on the complex interaction of S3-S4 and other membrane segments.

In the case of Kv channels, the activation process is complex and often facilitates transition into an inactivated state. Inactivated channels are not closed but do not carry any current due to a segment of the cytosolic region of the channel "plugging" the inner vestibule of the pore. The first 20 amino acids of the amino terminus form the inactivation gate for fast inactivation (215, 248). The positively charged inactivation gate swings toward the cytoplasmic face of the pore and binds to a negatively charged receptor region in the S4-S5 loop which occludes the pore and prevents K⁺ permeation. Interestingly, the inactivation ball can also be formed by soluble cytosolic accessory () subunits (454).

4. K⁺ channels are heteromultimeric complexes

The basic structure of all K⁺ channels is a tetramer of membrane-spanning subunits with a central pore. The subunits forming the tetramer may be the same protein (homotetramer) or two different proteins (heterotetramer) (see Table 1). For example, certain Kir channels consist of identical pore-forming subunits (Kir1.1 or ROMK) while others contain two different proteins (e.g., Kir4.1 and Kir5.1). In addition, many K⁺ channels exist as heteromeric assemblies of the pore tetramers with accessory proteins. Kv channels are complexes consisting of four -subunits that form the pore and cytosolic -subunits that modulate channel inactivation or other channel functions. In addition, calmodulin is complexed with the Ca²⁺-activated small-conductance K⁺ (SK) channel and through this interaction mediates calcium sensing (493). Moreover, some Kir channel tetramers may form heteromeric complexes with an ATP binding cassette (ABC) protein, like the cystic fibrosis transmembrane conductance regulator (CFTR), or the glibenclamide (sulfonylurea)-binding protein SUR2A/B.

The tetrameric nature of the K⁺ channel pore has been demonstrated using site-directed mutagenesis and X-ray crystallography (70, 233, 319, 447, 464, 502). -Subunits can coassemble to form either homo- or heteromultimers. Some of the structural elements that control tetrameric assembly have been identified in both Kv (628) and Kir (634) channels. At the amino terminus of Kv channels, the T1 domain is a conserved region 110 amino acid long proximal to S1 that mediates heteromultimeric interactions of Kv channels. The structure of T1 from Kv1.1 channels has been elucidated (189, 382). It is believed that active channels have four T1 domains that hang directly under the cytoplasmic mouth of the pore. They form a sort of platform, 20 away form the pore, connected to S1 by thin protein bands. K⁺ may access or leave the channel pore sideways, through 20--long openings defined by that T1 domain.

The electrophysiological properties of cloned subunits forming the tetrameric pore may not always match those known for these channels in native tissues (see Table 2). In general, this is due to pore-forming subunits interacting with accessory proteins in native cells thatmodify their kinetic and pharmacological properties. Furthermore, accessory proteins often modulate the levels of expression of pore-forming tetramers. These latter interactions with -subunits have been well-studied for Kv channels. For example, the Kv genes encode soluble proteins (367–404 amino acids), related to the NAD(P)H-dependent oxidoreductase superfamily, that interact with many Kv -subunits (442). The KChIP genes encode calcium sensors that modify the expression level and kinetic properties of Kv4.2 and Kv4.3 (297). The KChAP proteins act as chaperone for Kv proteins such as Kv1.3, Kv2.1, Kv2.2, and Kv4.3 (297, 617). The MinK-related proteins (MiRPs) also interact with Kv channels (1–3, 27, 471). KCNA4B encodes a soluble protein (141 amino acids) with limited similarity to the NAD(P)H-dependent oxidoreductase superfamily (552). KCNA4B binds to the carboxy terminus of KCNA10, increases KCNA10 current expression by almost threefold, and also alters its sensitivity to cAMP.

	II. FUNCTION AND REGULATION OF POTASSIUM CHANNELS IN THE KIDNEY

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1. K⁺ channels along the nephron

A) FUNCTION OF K⁺ CHANNELS IN PROXIMAL TUBULE.  The v0ast majority of filtered K⁺ is reabsorbed passively along the proximal tubule (164). Two mechanisms, passive diffusion and Na⁺-dependent solvent drag of tubule fluid, have been shown to mediate K⁺ retrieval from the filtrate. There is at present no convincing evidence that K⁺ reabsorption involves transcellular movement or K⁺ channels; rather, the bulk of K⁺ reabsorption is thought to proceed via paracellular pathways (612).

K⁺ channels in the proximal tubule serve at least three important functions (Fig. 2; Refs. 164, 168, 498). First, they participate in generating the cell negative membrane potential. Since several membrane transport processes are electrogenic, alteration in cell membrane potential can affect the Na⁺-coupled transport of molecules including glucose, phosphate, amino acids, and bicarbonate. Relevant examples include Na⁺-coupled phosphate, sulfate, and glucose entry into cells, transport processes which have been shown to be sensitive to membrane depolarization. In the basolateral membrane, Cl^– diffusion, Na⁺-coupled HCO₃^– cotransporter, and Na⁺/Ca²⁺ exchanger are affected by changes in basolateral membrane potential. Second, K⁺ channels in the proximal tubule are involved in regulating cell volume. For instance, stimulation of Na⁺-dependent carriers such as Na⁺-glucose cotransporters is expected to increase intracellular Na⁺ and cell volume. Such cell swelling activates both apical and basolateral K⁺ channels and increases K⁺ loss, restoring the cell volume. Third, K⁺ channels are responsible for K⁺ recycling across the basolateral membranes where they prevent K⁺ depletion in unstirred fluid layers in the complex system of interstitial spaces adjacent to the basolateral membrane.

In addition to the above-mentioned physiological function, ATP-sensitive K⁺ channels may be involved in hypoxic-induced renal injury (452). It was demonstrated in this latter study that inhibition of K⁺ channels in the proximal tubule by the sulfonylurea glibenclamide attenuates hypoxic injury.

 I) K⁺ channels in the basolateral membrane of mammalian proximal tubule.  Figure 5 and Table 2 summarize the biophysical properties of, and factors known to regulate, K⁺ channels in the basolateral membrane of the proximal tubule. Three types of K⁺ channels with single-channel conductances of 12 pS (405), 36–41 pS (171, 172), and 50–60 pS (35, 94, 172, 426, 441, 451, 498, 529, 563) have been observed in the basolateral membrane of the rabbit proximal tubule. An important feature of the 50- to 60-pS K⁺ channel is its sensitivity to inhibition by millimolar concentrations of ATP (35, 221). This may link (356) the function of this K⁺ channel to changes in basolateral Na⁺-K⁺-ATPase activity (also see Fig. 2). Stimulating the apical Na⁺-glucose cotransporter augments Na⁺ influx, which subsequently causes increased Na⁺-K⁺-ATPase activity in the basolateral membrane. Such enhanced Na⁺-K⁺-ATPase activity is expected to increase the hydrolysis of ATP and to lower intracellular ATP concentrations. With the decrease in intracellular ATP, ATP-sensitive K⁺ channels are relieved from ATP block, resulting in an increase in K⁺ recycling. In this way, the turnover rate of Na⁺-K⁺-ATPase can be tightly linked to the activity of basolateral K⁺ channels. This hypothesis is supported by several observations: 1) adding luminal glucose and alanine decreases the intracellular ATP concentrations and simultaneously increases basolateral K⁺ channel activity (563); 2) sulfonylurea agents, inhibitors of the ATP-sensitive K⁺ channels, also abolish the effect of stimulating Na⁺ transport on channel activity (563); and 3) inhibition of Na⁺-K⁺-ATPase increases intracellular ATP concentrations and inhibits basolateral K⁺ channel activity in proximal tubule cell (221).

View larger version (19K): [in this window] [in a new window]   FIG. 5. A model of proximal tubule cell illustrating the location and regulation of K+ channels. The single-channel conductance of the K+ channels is indicated. The luminal Na+-dependent glucose and other amino acid transporters are indicated by Na/S. The basolateral Na+-K+-ATPase is also shown.

The molecular nature of the ATP-sensitive K⁺ channel in the proximal tubule has been explored. Kir6.1 mRNA is expressed in the proximal tubule (405; see Fig. 3 and Table 1). Moreover, coexpressing Kir6.1 with a sulfonylurea receptor (SUR2A/2B) in oocytes results in an ATP-sensitive K⁺ channel that shares with the native basolateral K⁺ channels inhibition by taurine and glibenclamide (405). Therefore, it has been suggested that Kir6.1 is an important part of the basolateral 50- to 60-pS K⁺ channel. More discussion regarding the molecular nature of renal ATP-sensitive K⁺ channels can be found in section IVB3.

 II) K⁺ channels in the apical membrane of the mammalian proximal tubule.  Ca²⁺-activated large-conductance (200–300 pS; see Figs. 3, 4B, and 5; Table 2) or maxi-K⁺ channels have been identified in the apical membrane of cultured proximal tubule cells (42, 204, 205, 262, 380, 548) and in the brush border of rabbit proximal tubules (666). These channels are activated by mechanical stretch and membrane depolarization. However, since the channel open probability is very low, it is unlikely that these maxi-K⁺ channels contribute significantly to the apical K⁺ conductance under normal circumstances in native cells. However, it may play a role in stabilization of the apical membrane potential following stimulation of Na⁺-coupled glucose and amino acid transporters, which tend to depolarize the apical membrane. 33- and 63-pS K⁺ channels have also been found in the apical membrane of the proximal tubule (171; see Figs. 3 and 5; Table 2), although their physiological roles are unknown. A 42-pS ATP-regulated and pH-sensitive inwardly rectifying K⁺ channel has also been identified in the apical membrane of human cultured proximal tubule cells (395).

Molecular cloning has identified several types of voltage-gated K⁺ channels (638) and Ca²⁺-dependent maxi-K⁺ channels (193, 388) expressed in proximal tubules. Two voltage-gated K⁺ channels (e.g., KCNQ1, KCNE1, and KCNA10) have been localized to the apical membrane of proximal tubules (Fig. 3; Table 1; Ref. 569). These voltage-gated K⁺ channels may also be involved in stabilization of the apical membrane potential. The properties and potential roles of these voltage-gated K⁺ channels in the regulation of proximal tubule epithelial transport are discussed in section IV. The Ca²⁺-dependent maxi-K⁺ channel rbslo1 was cloned from rabbit renal cells and has homology to mslo; however, it is not clear whether rbslo1 transcripts are expressed in the rabbit proximal tubule (1 of 8 tubules gave a PCR signal; Ref. 388).

 III) K⁺ channels in the basolateral membrane of the amphibian proximal tubule.  At least two inward-rectifying K⁺ channels (27–30 pS, 47–50 pS) have been identified in the basolateral membrane of the proximal tubule of the amphibian kidney (Fig. 5 and Table 2; Refs. 218, 261, 367, 397, 397, 468, 566). The activity of the 27- to 30-pS K⁺ channel is increased by depolarization and membrane stretch (218, 260) and inhibited by ATP (367). In contrast, the activity of the 47- to 50-pS K⁺ channel is stimulated by hyperpolarization (261, 468). This feature suggests the possibility of metabolic coupling of the activity of Na⁺-K⁺-ATPase to that of basolateral K⁺ channels.

 IV) K⁺ channels in the apical membrane of amphibian proximal tubule.  A Ca²⁺-activated maxi-K⁺ channel has been identified in the apical membrane of the amphibian proximal tubule (147) and found to be activated by depolarization and membrane stretch. In contrast to the behavior of the basolateral stretch-activated K⁺ channels, the effect of mechanical stimulation on channel activity may be indirect and mediated by an increase in intracellular Ca²⁺. This view is based on the observation that membrane stretch activates a Ca²⁺-permeant nonselective cation channel in the apical membrane of proximal tubules (147, 260, 466, 467).

B) FUNCTION OF K⁺ CHANNELS IN THE TAL.  The TAL reabsorbs not only 20–25% of the filtered Na⁺ load but also plays an important role in reabsorption of divalent cations such as Mg²⁺ and Ca²⁺. K⁺ channels in the TAL are important in generating the cell membrane potential, apical K⁺ recycling coupled to K⁺ entry via Na⁺-K⁺-2Cl^– cotransport, regulating cell volume, and basolateral K⁺ recycling coupled to the activity of Na⁺-K⁺-ATPase (see Figs. 2 and 6).

View larger version (25K): [in this window] [in a new window]   FIG. 6. A model of TAL cell showing K+ channels and their regulation. The single-channel conductance of the K+ channels is indicated. The luminal Na+-K+-Cl– cotransporter and the basolateral Na+-K+-ATPase are shown. 20-HETE, 20-hydroxyeicosatetraenoic acid; PKC, protein kinase C; PKA, protein kinase A; PKG, cGMP-dependent protein kinase; PTK, protein tyrosine kinase; CO, carbon monoxide; AA, arachidonic acid.

 II) K⁺ channels in the apical membrane of mammalian TAL.  K⁺ channels play a key role in K⁺ recycling across the apical membrane of the TAL. Their importance is underscored by the observation that inhibition of apical K⁺ recycling impairs NaCl reabsorption in the loop of Henle (164, 180, 195, 593). Several functions are served by K⁺ recycling (see Fig. 6). First, K⁺ recycling hyperpolarizes the cell membrane potential, which is an essential factor for Cl^– diffusion across the basolateral membrane. Second, K⁺ recycling across the apical membrane, coupled to Cl^– exit across basolateral membranes, generates the lumen-positive transepithelial potential that is the main driving force for paracellular Na⁺, Ca²⁺, and Mg²⁺ transport. Third, K⁺ recycling maintains an adequate supply of K⁺ for the Na⁺-K⁺-2Cl^– cotransporter in the cortical TAL.

Three types of K⁺ channels, a low-conductance (30–35 pS), an intermediate-conductance (70–72 pS), and a large-conductance (140–200 pS) maxi-K⁺ channel, have been observed in the apical membrane of the TAL (Fig. 6 and Table 2; Refs. 49, 94, 188, 589, 599). Patch-clamp studies have demonstrated that both the low- and intermediate-conductance K⁺ channels contribute to the apical K⁺ conductance and to K⁺ recycling. Both the large apical membrane conductance and K⁺ recycling can be accounted for by the high open probabilities and channel densities (593). For example, the 70-pS K⁺ channel has been estimated to contribute up to 75% of the total apical K⁺ conductance of the TAL in rat maintained on a high-K⁺ diet (604). In contrast, the open probability of the maxi-K⁺ channel is very low under normal conditions in native cells and therefore is unlikely to play a significant role in apical K⁺ recycling (188, 545). However, the maxi-K⁺ channel in TAL cell culture can be activated by membrane stretch (545) and thus may be involved in regulating cell volume. It has not been explored whether increased flow rate in the TAL could stimulate maxi-K⁺ channels as has been described for principal cells in the cortical collecting duct (CCD) (620). Such a mechanism has been implicated in the CCD (546, 620) and, therefore, could provide an additional K⁺ recycling mechanism in the TAL.

The molecular nature of the low-conductance, 35-pS K⁺ channel is established to be ROMK (Kir1.1; Fig. 2 and Table 1; see sect. III; Refs. 342, 593). The ROMK-knockout mouse exhibits a Bartter's-like phenotype (333, 342), and these mice lack expression of both the 35- and 70-pS K⁺ channels in the TAL (342). Thus it is possible that ROMK may also be involved in forming the 70-pS K⁺ channel.

 III) Regulation of K⁺ channels in the TAL.  Figure 6 provides a summary of the factors regulating apical and basolateral K⁺ channels in the mammalian TAL (166, 593).

 A) The apical 35-pS K⁺ channel.  Vasopressin increases the activity of this K⁺ channel via a cAMP-dependent pathway, and the channel can be directly stimulated by cAMP-dependent protein kinase (PKA) in the absence of hormone (451, 589). Since vasopressin also stimulates Na⁺ reabsorption in the TAL (180, 196, 197), it is possible that at least part of the vasopressin effect is mediated by increasing apical K⁺ recycling. Consistent with this notion, the arginine vasopressin analog 1-deamino-(8-D-arginine)-vasopressin (DDAVP) increases Kir1.1 (ROMK) abundance in the TAL of the Brattleboro rat (125).

The 35-pS K⁺ channels are inhibited by millimolar intracellular ATP and by acidic pH (48, 589). The former may be involved in metabolic sensing to coordinate apical and basolateral potassium fluxes as demonstrated in proximal tubule (563, 613) and collecting duct principal cells (602). pH sensing by the 35-pS K⁺ channel may serve to modulate apical K⁺ conductance during acid-base disturbances (pH reductions are inhibitory) and also may be involved in the altering of channel activity by other factors [e.g., phosphatidylinositol 4,5-bisphosphate (PIP₂); see sect. III]. In addition, modulating the activity of the Na⁺-K⁺-2Cl^– cotransporter may alter intracellular pH and affect apical K⁺ conductance as in the amphibian diluting segment (93).

Since ROMK encodes the 35-pS K⁺ channel, modulation of its expression by hormones or dietary factors provides information about regulation of this low-conductance channel. Increase in glucocorticoids, protein, or sodium intake enhances ROMK expression in the TAL while low sodium intake reduces channel expression (125, 158). In addition, increases in osmolality also enhance ROMK expression ib the medullary TAL (158).

 B) The apical 70-pS K⁺ channel.  The 70-pS K⁺ channel is also stimulated by PKA and inhibited by millimolar intracellular ATP (49, 589, 599). Moreover, two eicosanoids, 20-hydroxyeicosatetraenoic acid (20-HETE) and PGE₂, play important roles in regulating the activity of this K⁺ channel. In the medullary TAL (mTAL), 20-HETE is the major metabolite of cytochrome P-450 -oxidation of arachidonic acid, whereas PGE₂ is the major product of cyclooxygenase-dependent metabolism of arachidonic acid in the mTAL (75, 76, 369). Addition of arachidonic acid inhibits the 70-pS K⁺ channel (596), and this effect can be mimicked by 20-HETE. The latter eicosanoid has also been shown to inhibit the Na⁺-K⁺-2Cl^– cotransporter (134). Reduction in the activity of the 70-pS K⁺ channel may be an additional mechanism by which 20-HETE lowers the activity of the Na⁺-K⁺-2Cl^– cotransporter via a decrease in apical K⁺ recycling. At low concentrations, PGE₂ inhibits the 70-pS K⁺ channels by reducing cAMP level, whereas high concentrations of PGE₂ directly inhibit the channel activity by stimulating protein kinase C (PKC) (328).

It has been suggested that extracellular Ca²⁺-mediated stimulation of the basolateral G protein-coupled Ca²⁺-sensing receptor (CaSR) provides a major mechanism for regulation of NaCl and Ca²⁺/Mg²⁺ transport by the TAL (66). Increased blood/interstitial concentrations of Ca²⁺, Mg²⁺, or aminoglycoside antibiotics (e.g., neomycin) activates the CaSR resulting in reductions in both divalent mineral and NaCl reabsorption (99, 241, 242, 446, 537, 538). As a consequence, divalent mineral excretion is enhanced, and countercurrent multiplication and urinary concentrating ability are reduced. Ca²⁺-mediated activation of the CaSR in the TAL reduces cAMP production by adenylate cyclase (probably type 6) and enhances cAMP destruction by phosphodiesterases (PDE) (241, 242). This CaSR-mediated reduction in cAMP generation would reduce the activity of both the 70- and 35-pS K⁺ channels (Fig. 6). In addition, stimulating the CaSR in the TAL significantly increases 20-HETE production, which inhibits the 70-pS K⁺ channel (604). Since the CaSR is half-maximally activated at the normal plasma concentration of Ca²⁺, the CaSR could account for the basal production of 20-HETE. Thus CaSR activation can inhibit both types of K⁺ channels involved in apical K⁺ recycling, consistent with reduction in NaCl and Ca²⁺/Mg²⁺ transport by the TAL. The potent effect of CaSR activation on apical K⁺ channels in the TAL has been recently supported by the observations that certain activating (gain-of-function) mutations of the human CaSR gene produce a Bartter's-like phenotype (573, 609).

It has also been suggested that 20-HETE may mediate the low K⁺ intake-induced decrease in the activity of the 70-pS K⁺ channel, since the concentration of this eicosanoid is four times higher in the mTAL from rats on a K⁺-deficient diet than in those from animals on a high-K⁺ diet (185). Channel open probability is significantly diminished in mTAL harvested from rats on a K⁺-deficient diet (185). In contrast, inhibiting cytochrome P-450 -oxidation increases channel activity to an extent similar to that observed in the high K⁺ adapted animals (185).

The apical 70-pS K⁺ channel is activated by two gases, nitric oxide (NO) and carbon monoxide (CO) (327, 346). Inhibition of NO synthase (NOS) attenuates, whereas addition of NO donors increases, the activity of the 70-pS K⁺ channel. The effect of NO is mediated by a cGMP-dependent pathway because the effect of NOS inhibitors can be reversed by membrane-permeant cGMP analogs (346). The mechanism by which CO regulates the 70-pS K⁺ channel is unlikely to involve cGMP because CO effects can also be observed in excised patches (327).

 C) The basolateral K⁺ channel.  Application of arachidonic acid (AA) reduces the activity of the 41-pS K⁺ channel, and this effect of AA is abolished by blocking the cytochrome P-450 metabolic pathway (see Fig. 6). Thus the effect of AA may also be mediated by 20-HETE (183). Given that basolateral K⁺ channels play an important role in maintaining the driving force for Cl^– exit across the basolateral membrane, inhibition of basolateral K⁺ channels is expected to decrease Cl^– transport.

 IV) K⁺ channels in the amphibian diluting segment.  An inward-rectifying K⁺ channel with a conductance of 25–31 pS has been identified in the apical membrane of the frog diluting segment which shares the properties of the TAL (223–225). This channel is activated by increasing cell pH from 7.4 to 8.2 and inhibited by acidic pH (6.6). Furosemide, which inhibits Na⁺-K⁺-2Cl^– cotransport and increases intracellular pH, activates the apical K⁺ channel (93). With the use of isolated cells from the frog diluting segment, three types of K⁺ channels with differing conductances have been found (24, 45, and 59 pS; Ref. 594). However, their membrane location is not known. Of interest is the finding that aldosterone stimulates the 45-pS K⁺ channel that is sensitive to changes in intracellular pH (594). Because aldosterone raises the cell pH in frog diluting segment, it is possible that the effect of aldosterone on the 45-pS K⁺ channel results from increasing cell pH (594).

C) FUNCTION OF K⁺ CHANNELS IN THE MAMMALIAN DISTAL CONVOLUTED TUBULE.  The distal convoluted tubule (DCT) plays an important role in the regulation of Na⁺, Mg²⁺, and Ca²⁺ transport (26, 95, 98). However, only a few studies have assessed the properties of K⁺ channels in this nephron segment. A 48- to 60-pS K⁺ channel has been observed in the basolateral membrane of the rabbit distal tubule (547). In addition, an inwardly rectifying K⁺ channel with inward slope conductance of 37 pS has been observed in the cultured mouse distal tubule cells, and this channel is inhibited by acidic pH (337). Specific roles of these channels in distal tubule function are unknown.

D) FUNCTION OF K⁺ CHANNELS IN THE COLLECTING DUCT.  The CCD plays a key role in the regulated secretion of K⁺ (164, 168; see Figs. 1, 2, and 7). K⁺ secretion occurs in principal cells by a two-step process: active entry of K⁺ across the basolateral membrane mediated by Na⁺-K⁺-ATPase, followed by passive diffusion across the luminal membrane via apical K⁺ channels along a favorable electrochemical gradient (164). In addition to playing an important role in the secretion of K⁺, it has been suggested that apical K⁺ channels are also involved in modulating the process of reabsorption of K⁺ that occurs in cells of the outer medullary collecting duct (OMCD). In these cells, K⁺ channels are coupled to the activity of K⁺/H⁺ exchange (H⁺-K⁺-ATPase); channel activity is high during K⁺ repletion but decreases sharply during K⁺ depletion (653, 654). Thus variable apical recycling of K⁺, in concert with regulated basolateral K⁺ channel activity, could be an important mechanism modulating the efficacy of active K⁺/H⁺ exchange.

View larger version (25K): [in this window] [in a new window]   FIG. 7. A model illustrating the K+ channels and their regulation in principal cells from the CCD. The luminal epithelial Na+ channel (ENaC) and the basolateral Na+-K+-ATPase are shown. PKC, protein kinase C; PKA, protein kinase A; PKG, cGMP-dependent protein kinase; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; CaMK II, calcium and calmodulin-dependent kinase II; CO, carbon monoxide; NO, nitric oxide; AA, arachidonic acid; PP2A, protein phosphatase 2A; ADH, antidiuretic hormone.

 I) K⁺ channels in the apical membrane of mammalian CCD.  Low-conductance (35 pS), maxi-K⁺ (140 pS), and voltage-gated (10–16 pS) channels have been identified in the apical membrane of the CCD (Fig. 7; Refs. 152, 153, 209, 220, 324, 478, 483, 525, 598). The low-conductance and voltage-gated channels are restricted to principal cells (153, 598). However, the maxi-K⁺ channel has been observed in both principal and intercalated cells, although it is mostly expressed in intercalated cells (152, 219, 220, 418).

The 35-pS K⁺ channel mediates the bulk of K⁺ secretion in the CCD under normal conditions when distal flow is not high (152, 153). The 35-pS K⁺ channel is encoded by Kir1.1 (ROMK; Refs. 168, 213, 342, 652). This was demonstrated by the absence of this 35-pS K⁺ channel in principal cells in the ROMK knockout mouse (342). The structure, function, and regulation of the ROMK channel are discussed in section III. With high luminal flow rates, the maxi-K⁺ channel may contribute significantly to net K⁺ secretion (546, 620). Moreover, maxi-K⁺ channels do not contribute significantly to the apical K⁺ conductance under normal physiological conditions because its open probability is very low (152). The maxi-K⁺ channel is encoded by rbsol1, a mslo homolog (388). It is of interest that maxi-K⁺ channels in the perfused Ambystoma collecting duct appear to mediate K⁺ secretion during exposure to high concentrations of KCl (526, 527). Activity of the voltage-gated channels has been shown to be increased by membrane depolarization and may be encoded by Kv1.3, although the membrane localization of this channel is unclear. The voltage-gated K⁺ channel may stabilize the membrane potential whenever high rates of luminal Na⁺ tend to depolarize this membrane (106).

 II) Regulation of apical K⁺ channels.  Figure 7 illustrates the known mechanisms by which the apical 35-pS, maxi-K⁺, and voltage-gated K⁺ channels are regulated in the CCD.

 A) The maxi-K⁺ channel.  The Ca²⁺-activated maxi-K⁺ channel is sensitive to TEA, ATP, and acidic pH (152, 207, 209, 219). The sensitivity to ATP and pH depends on the cell Ca²⁺; in the presence of 1 mM Ca²⁺, neither ATP nor acidic pH can inhibit the channel activity, whereas 1 mM ATP and acidic pH decrease the K⁺ channel activity in the presence of 1 µM Ca²⁺ (207). The maxi-K⁺ channels are also activated by hypotonic cell swelling (209). In addition, increases in flow rate stimulate K⁺ secretion (131, 359, 360), which is associated with an increase in intracellular Ca²⁺ (621). It has been suggested that maxi-K⁺ channels mediate this Ca²⁺-associated, flow-dependent K⁺ secretion. This is based on the observation that TEA (152) or charybdotoxin (546) inhibits K⁺ secretion with high luminal flow rates. TEA does not alter K⁺ secretion during low flow rates. Moreover, deletion of the gene encoding the ₁-accessory subunit of maxi-K⁺ channels impairs K⁺ excretion following acute volume expansion, an effect consistent with a role for these channels in K⁺ excretion during high rates of distal tubule flow rate (440). The mechanism, by which a high flow rate increases intracellular Ca²⁺ and presumably maxi-K⁺ channel activity, is not completely understood but may be related to an increase in cell Ca²⁺ (546). One hypothesis for coupling flow to increases in intracellular Ca²⁺ in principal cells involves flow-dependent deformations of the central cilium (443, 620). Deformation of the cilium increases intracellular Ca²⁺ through opening of mechanically sensitive channels that probably reside in the cilium or its base. This influx of Ca²⁺ is followed by Ca²⁺ release from inositol 1,4,5-trisphosphate (IP₃)-sensitive stores. Therefore, increase in intracellular Ca²⁺ would activate maxi-K⁺ channels and increase in K⁺ secretion. Intercalated cells in the CCD lack a central cilium; however, increases in flow rate still raise intracellular Ca²⁺ in these cells. Therefore, a mechanism other than cilium deformation is likely to be involved in mediating the effect of increasing flow rate on intracellular Ca²⁺ in intercalated cells.

 B) The 35-pS K⁺ channel.  The activity of the 35-pS K⁺ channel is regulated by a large number of factors including nucleotides, phosphatidylinositol phosphates, cytosolic pH, kinases, phosphatases, and arachidonic acid. These factors regulate both channel gating and density in the membrane (see Fig. 7). The activity state of the channel is complexly determined not only by these individual factors but also by the interplay of multiple factors that influence each other.

The 35-pS K⁺ channel is regulated by both cytosolic and extracellular ATP. The channel is inhibited by millimolar concentrations of cytosolic Mg-ATP (592, 598), and channel sensitivity to ATP could play a role in linking its activity to Na⁺-K⁺-ATPase (221, 563). External ATP has also been shown to inhibit the 35-pS K⁺ channel via purinergic receptor-mediated effects (340). P2 type purinergic receptors are present on both apical and basolateral membranes of principal cells (304). Stimulating P2Y2 purinergic receptors increases cytosolic Ca²⁺ (340) which, in turn, enhances cGMP generation by activating Ca²⁺-regulated NOS (338). Increasing cGMP activates protein phosphatases and facilitates the dephosphorylation of K⁺ channels. Because the phosphorylation state of the channel in principal cells is an important determinant of its activity (350, 630), enhancing dephosphorylation would be expected to lower channel activity.

The 35-pS K⁺ channel in principal, as in TAL, cells is sensitive to changes in cell pH in the physiological range. Decreasing pH from 7.4 to 7.2 reduces channel activity by 50% (482, 598). K⁺ secretion is inhibited during acidosis and likely contributes to the effect of acidosis on K⁺ excretion (164).

Arachidonic acid inhibits the 35-pS K⁺ channel in the CCD (591). The effect of arachidonic acid is specific because other fatty acids such as lenoleic or palmic acid cannot mimic its effect. Moreover, the effect of arachidonic acid is not mediated by its metabolites since inhibiting cyclooxygenase, lipoxygenase, and cytochrome P-450 monooxygenase does not block the effect of arachidonic acid.

Ca²⁺-dependent signal transduction pathway kinases, such as PKC and calmodulin-dependent kinase II (CaMKII), inhibit the 35-pS K⁺ channel (290). Increasing intracellular Ca²⁺ reduces the activity of the 35-pS K⁺ channel, and this effect is blocked by inhibiting PKC or CaMKII and mimicked by adding the catalytic subunits of PKC and CaMKII (602).

PKA and Mg-ATP play important roles in regulating the activity of the 35-pS K⁺ channel. Modulation of kinases by application of cAMP or inhibition of phosphatases stimulates the channel phosphorylation and increases the number of the functional 35-pS K⁺ channel in the cell membrane (77</