Regulation of potassium (K) handling in the renal collecting duct

Abstract

This review provides an overview of the molecular mechanisms of K transport in the mammalian connecting tubule (CNT) and cortical collecting duct (CCD), both nephron segments responsible for the regulation of renal K secretion. Aldosterone and dietary K intake are two of the most important factors regulating K secretion in the CNT and CCD. Recently, angiotensin II (AngII) has also been shown to play a role in the regulation of K secretion. In addition, genetic and molecular biological approaches have further identified new mechanisms by which aldosterone and dietary K intake regulate K transport. Thus, the interaction between serum-glucocorticoid-induced kinase 1 (SGK1) and with-no-lysine kinase 4 (WNK4) plays a significant role in mediating the effect of aldosterone on ROMK (Kir1.1), an important apical K channel modulating K secretion. Recent evidence suggests that WNK1, mitogen-activated protein kinases such as P38, ERK, and Src family protein tyrosine kinase are involved in mediating the effect of low K intake on apical K secretory channels.

Overview of K transport mechanism in CNT and CCD

Maintaining plasma K within a narrow physiological range is essential for the function of neurons, cardiac myocytes, and skeletal muscles. The kidney plays a key role in regulating K excretion by completely filtering K in the glomerulus, reabsorbing K extensively along the proximal tubule and thick ascending limb, secreting K in the connecting tubule (CNT) and cortical collecting duct (CCD), and reabsorbing K in outer medullary collecting duct (OMCD). Two morphological distinct cells, principal cell (PC) and intercalated cell (IC), are present in the CNT and CCD [29, 75], and it is generally accepted that PC and IC are responsible for K secretion and for K absorption, respectively [27, 30]. Figure 1 is a cell model illustrating the K transport mechanism under control conditions (normal K intake) in both PC and IC in the CCD [29, 74, 75]. K secretion takes place by a two-step process: K enters the cell via the basolateral Na,K-ATPase and is secreted into the lumen through apical K channels along a favorable electrochemical gradient [76, 101]. K absorption is achieved by K entering the cell across the apical membrane through a luminal H,K-ATPase and leaving the cell across the basolateral membrane along a favorable K electrochemical gradient [19, 21, 33]. Although H,K-ATPase is mainly expressed in IC in collecting duct, colonic H,K-ATPase has been shown to be expressed in PC of connecting tubule and the CCD [115].

Fig. 1.

A model of principal cell (PC) and intercalated cell (IC) illustrates the K transport under control conditions (normal K intake)

Luminal Na transport provides normally an important driving force for K secretion [97], but recent evidence has shown that K secretion may continue when luminal Na transport is compromised. As demonstrated in microperfused rabbit CCD, K secretion may continue, albeit at a reduced rate, in the absence of luminal Na [69]. Since inhibition of basolateral Na/H exchanger significantly decreases such K secretion in the absence of luminal Na, it is most likely that Na recycling across the basolateral membrane through Na/H exchange supplies enough Na to sustain the activity of Na,K-ATPase which is essential for K secretion.

In addition to entering PC by Na,K-ATPase, K can also gain cell access across the basolateral membrane via K channels in the CCD provided the basolateral membrane hyperpolarizes to exceed the K equilibrium potential. This may occur as consequence of mineralocorticoid-induced stimulation of Na,K-ATPase [92]. Three types of K channels have been identified in the basolateral membrane of the CCD and shown to be activated by cGMP-dependent pathways [32, 37, 120, 123]. The regulation of Na,K-ATPase in the CCD has been covered in two review articles [24, 116] and will not be further discussed in the present review. Thus, this review paper is focused largely on the apical K transport mechanisms, especially apical K channels, in the CNT and CCD.

Apical K transport in the CNT and collecting duct

K channels

Several types of K channels including ROMK (Kir1.1), a Ca²⁺-activated big-conductance K channel (BK) and double-pore K channel, KCNK1, are expressed in the apical membrane of the CNT and CCD [25–27, 38, 78, 79, 95]. It is now well established that ROMK and BK channels are responsible for K secretion. In contrast, further experiments will be necessary to define the role of the two-pore K channels in K transport in the CNT or CCD. ROMK channels have similar biophysical properties and regulatory mechanism as that of the native small-conductance K (SK) channels identified in the mouse and rat CCD [35]. Thus, we terminate the SK as a ROMK-like SK channels in the present review. Since ROMK-like SK channels have a high open probability and are abundantly expressed in the apical membrane of the CNT and CCD under control conditions, ROMK channels are thought to play a major role for K secretion under normal dietary K intake [26, 31, 56, 93, 95, 100, 119]. However, when the tubule flow rate is high or dietary K intake increases [9, 94, 136], both BK channels and ROMK-like SK are involved in mediating K secretion.

ROMK

The ROMK channel [36] is a member of inwardly rectifying K (K_IR) channels [72] that are functionally characterized by high K selectivity and either weak or strong inward rectification. The information about ROMK structure is largely obtained from the X-ray crystallographic structure of a K channel from Streptomyces lividans [20], demonstrating that each contains two membrane spanning segments and cytoplasmic N and C termini with high homology to the pore-forming H5 segment of voltage-gated K channels. Moreover, Minor et al. [66] employed a yeast genetic screening technique to analyze the packing structure of the M1 and M2 domains. They suggest that M2 segments line the pore and are surrounded by M1 segments which also participate in subunit–subunit interactions in the tetrameric channel complex.

ROMK channels are pH-sensitive, and a decrease in cell pH from 7.4 to 7.0 completely inhibits channel activity (which is defined by NP_o, a product of channel number and open probability) [14, 85, 119]. Structure and function analysis have demonstrated that interaction between lysine residue 80 and alanine residue 177 on M2 domain is essential for the pH sensitivity of ROMK [85]. The native ROMK-like SK channel is sensitive to ATP [119], but ATP sensitivity is absent in ROMK channels expressed in oocytes. Several studies have demonstrated that ROMK channels may interact with CFTR or sulfonylurea receptor type 2B (SUR2B) and that such interaction is required for the ATP sensitivity [18, 59, 63]. ROMK channels have three putative PKA phosphorylation sites, and stimulation of PKA-induced phosphorylation increases ROMK channel activity [1, 64, 138]. This is achieved by either enhancing the insertion of ROMK channels into plasma membrane [73, 141] or by augmenting the effect of PIP2 [57] which has been shown to activate ROMK channels [40, 58]. Serum-glucocorticoid-induced kinase 1 (SGK1) also stimulates ROMK channel activity [143] by increasing the phosphorylation of a serine residue of the N terminus of ROMK (Ser 44 for ROMK1), a putative PKA phosphorylation site [140]. Thus, SGK1 stimulates the surface expression of ROMK1 channels through facilitating the export from endoplasmic reticulum. Recently, it has been suggested that SGK1 stimulates ROMK channels by phosphorylation of WNK4 [88]. The effect of PKC on ROMK channel is complex because PKC has both stimulatory and inhibitory effects [53, 144]. PKC-induced phosphorylation of ROMK channels is required for export of ROMK1 channels to the cell membrane [53]. However, stimulation of PKC inhibits ROMK channels by decreasing the sensitivity of ROMK channels to PIP2 [144]. ROMK1 channels are also a substrate for Src family protein tyrosine kinase (PTK) which increases the endocytosis of ROMK1 channels in the CCD by stimulation of tyrosine phosphorylation [54, 67].

Ca²⁺-activated BK

The BK channel is composed of a pore-forming α subunit (Slo 1) with six transmembrane segments and an accessory β subunit [60, 91]. BK channel activity has been detected at the apical membrane of both PC and IC of the CCD [25, 42]. Real-time polymerase chain reaction performed in the isolated single CCD has demonstrated that BK channel α, β2, and β4 subunits, but not β1, are expressed in rabbits fed a high K diet. Moreover, high K intake stimulates the transcription of BK channel β2 and 4 subunits [70]. In contrast, low Na intake, which increases aldosterone level, does not affect the messenger RNA level of BK α, β2, and β4 subunits in rabbit CCD, suggesting that aldosterone does not contribute to the regulation of BK channel expression [23]. BK channels are sensitive to cell pH and ATP at physiological Ca²⁺ levels [98, 99]. Their contribution to K secretion was initially deemed uncertain in light of their very low open probability in CCDs. Because such patch-clamp experiments were performed in split-open tubules with no fluid flow [25, 51], it is possible that in vivo BK channel activity may be higher. This possibility is suggested by two recent studies highlighting the role of BK channels in flow-stimulated K secretion in both the CNT [112] and the CCD [136]. The role of BK channel in flow-stimulated K secretion is also supported by the observation that an increase in flow failed to stimulate K secretion in the distal nephron in BK-α subunit knockout mice [87]. It is of interest that patch-clamp experiments have demonstrated high BK channel activity in IC [84]. Because IC has a low Na,K-ATPase activity, it is difficult to explain their possible contribution to significant rates of K secretion [10]. However, two lines of evidence suggest that BK channels in PC are involved in mediating renal K secretion: (1) BK channel activity in PC is increased in the CCD from rats on a high K diet and (2) BK channel activity is significantly augmented by inhibition of P38 and ERK, both of which are suppressed by HK intake [51]. Although BK channels play a role in flow-induced stimulation of K secretion, deletion of the BK channel α subunit does not affect the net K excretion in mice fed with high K. This suggests that HK-induced stimulation of ROMK channel expression and high plasma aldosterone level can compensate for deleting BK channels on K secretion [87].

KCl co-transport

The KCl co-transporter (KCC), most likely KCC1, has been shown to be expressed in the apical membrane of the distal nephron including CCD [52]. Several studies suggest a role for apical KCC in renal K secretion. A reduction in luminal Cl markedly increases K secretion in perfused rat distal tubules, a mixture of distal convoluted tubule, CNT, and initial CCD [22, 114]. This component of K secretion is not influenced by luminal Ba²⁺ or amiloride [22] and can be blocked by luminal inhibitors of KCl co-transporters [2]. These findings have been extended to the rabbit CCD where a decrease in luminal Cl from 112 to 5 mM increases K secretion by 48% [134]. A reduction in basolateral Cl also decreases K secretion without an effect on transepithelial voltage or Na transport. The direction of K flux can be reversed by a lumen-to-bath Cl gradient, resulting in K absorption. In perfused CCDs from rats treated with mineralocorticoid, vasopressin increases K secretion [96]. Since this increase in K secretion is resistant to luminal Ba²⁺, vasopressin may stimulate apical KCC in the distal tubule [2].

H,K-ATPase/K-ATPase

Two types of H,K-ATPase are expressed in the kidney: colonic H,K-ATPase, which is sensitive to both ouabain and Sch28080, and gastric H,K-ATPase, which is inhibited by Sch-28080 [6, 15, 21, 47, 103, 132]. Molecular cloning has revealed that colonic and gastric H,K-ATPase share 60–70% sequence homology and that gastric H,K-ATPase and colonic H,K-ATPase contain HKα1 (type I K-ATPase) and HKα2 (type III K-ATPase), respectively [39]. Several early studies have suggested that gastric H,K-ATPase was involved in renal K absorption from K-depleted animals [12, 28, 142]. It had been reported that Rb influx, an index of K transport, increased in the OMCD from rabbits on a K-deficient diet and that this effect was abolished by inhibition of gastric H,K-ATPase [147]. However, the late investigations have shown that colonic H,K-ATPase, rather than gastric H,K-ATPase, is mainly responsible for renal K reabsorption and that gastric H,K-ATPase is involved in mediating K-dependent proton secretion in collecting duct [39]. First, application of ouabain inhibited Sch-28080-sensitive Rb²⁺ absorption in OMCD [146]. Second, K restriction significantly increases the expression of type III K-ATPase [103]. Third, K-ATPase activity determined by ATP hydrolysis rate in the CCD and OMCD is not affected in HKα1 (−/−) mice on K-deficient diet, while no K-ATPase activity was detected in HKα2(−/−) mice on K-deficient diet [17]. However, deletion of colonic H,K-ATPase does not display the renal phenotype even in mice fed on K-deficient diet [65], suggesting kidney has an alternative mechanism to reabsorb K in HKα2(−/−) mice. Moreover, immunostaining has shown that colonic H,K-ATPase is also detected in the apical membrane of PC of the CCD during K restriction, suggesting that PC is also involved in K reabsorption [33].

Regulation of K excretion by K diet

K transport in the CNT and CCD is regulated by hormones such as aldosterone and dietary K intake. Dietary K intake plays a key role in the regulation of renal K secretion: High K intake stimulates, whereas low K intake decreases renal K secretion [79, 83, 121]. Recently, several new mechanisms by which K intake regulates apical secretory K channels in the CCD have been reported. They include WNK, SGK1, and CYP-epoxygenase-dependent metabolites of arachidonic acid.

Effect of high dietary K intake

High K-intake-induced stimulation of renal K secretion is mediated by both aldosterone-dependent and an aldosterone-independent mechanism. Figure 2 is a cell model illustrating the current understanding of the effects of high K intake on K transport in PC and IC. HK intake stimulates aldosterone secretion which augments activity of both Na,K-ATPase and ENaC [24, 80, 89, 109, 116, 131]. Increased ENaC activity augments the driving force for K exit across the apical membrane of the CNT and CCD, whereas high Na,K-ATPase activity stimulates K secretion by increasing K uptake across the basolateral membrane. Mineralocorticoid receptor (MR) knockout mice have severe hyperkalemia and hyponatremia, underscoring the importance of aldosterone in the regulation of renal K secretion [11]. Moreover, high K intake significantly increases the activity of ROMK-like SK and BK channels, at least in part, by stimulating the expression of BK channel α subunit in the CCD [51, 70, 121]. However, the high K-intake-induced stimulation of ROMK-like SK channel activity may also require the involvement of factors other than aldosterone because infusion of aldosterone or application of low Na diet, a maneuver which increases circulated aldosterone level, fails to mimic the effect of high K intake [81, 82]. The notion that high K intake increases K secretion by an aldosterone-independent mechanism is also suggested by the observation that high K intake continues, albeit at a reduced rate, to stimulate K secretion in the isolated perfusion CCD from adrenalectomized rabbit [68]. HK intake inhibits K reabsorption not only by decreasing colonic H,K-ATPase expression [39] but also through enhancing apical K channel activity in IC [51]. This may indirectly affect the activity of H,K-ATPase by enhancing K recycling across the apical membrane, thereby diminishing net K reabsorption. This review is supported by the observation that inhibition of apical K channels in IC suppresses the H,K-ATPase activity (measured by proton extrusion) in K-repleted animals but has no effect in the K-restricted animals [145]. Although apical K channels in the IC play a role in the regulation of H, K-ATPase activity, their regulation mechanism has not yet been extensively explored. In contrast, advance in molecular biology has identified new signaling pathway regulating the apical K channels in PC. Figure 3 is a scheme illustrating the current understanding about mechanisms by which high K intake regulates apical K channels in the CCD through aldosterone-dependent and -independent signaling pathways. First, high K intake is expected to abolish the inhibitory effect of WNK4 on ROMK channels through aldosterone and SGK1 pathway. Second, high K intake suppresses renin-AngII signaling pathway which could decrease both ROMK and BK channel activity by a mitogen-activated protein kinases (MAPK)-dependent mechanism [7, 51]. Third, high K intake stimulates CYP epoxygenase activity and increases 11,12-EET production which stimulates BK channel activity [111]. However, the first possibility that aldosterone may stimulate ROMK channel by SGK1-dependent mechanism is only a speculation and needs to be examined in the future study.

Fig. 2.

A cell scheme illustrating the mechanism by which high K intake stimulates K secretion in the CCD by an aldosterone-dependent and -independent mechanisms. Solid arrow and dotted arrow indicate enhanced and diminished effect, respectively

Fig. 3.

A scheme showing the role of different signaling pathways in mediating the effect of high K intake on ROMK and BK channels in the CCD. The circle with x indicates the inhibition of a particular signaling pathway

WNKs and KS-WNK1

WNKs belong to a family of serine/threonine protein kinases. Four mammalian WNKs have been identified [46], of which WNK1, 3, and 4 are expressed in the CCD [45, 46, 50] and play an important role in the regulation of ROMK channels [16, 44, 48, 50, 88, 117]. Co-expression of WNK1, 3, and 4 inhibits the ROMK channel activity in Xenopus oocytes, and the effect of WNKs on ROMK is mediated by stimulation of clathrin-dependent endocytosis [44]. Recently, it has also been demonstrated that intersectin, a scaffold protein containing two Eps15 homology domains and four or five tandem SH3 domains, is required for the interaction between WNK4 and clathrin [34]. In addition, 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 CCD [77]. Unlike the long form of WNK1 which inhibits ROMK channels [16], KS-WNK1 lacks kinase activity and does not block ROMK channels. Moreover, KS-WNK1 can antagonize the inhibitory effect of WNK1 [48, 117]. It has been reported that high K intake increases the expression of KS-WNK1 and accordingly attenuates the inhibitory effect of WNK1 on ROMK channels [48, 117]. Thus, the alteration of the ratio between long and short form of WNK1 may be an important mechanism by which high K intake stimulates ROMK channel activity. Moreover, WNK1 has also been reported to stimulate SGK1 through PI3 kinase [137]. Because high K intake is expected to increase SGK1, which stimulates K secretion [113], WNK1-mediated activation of SGK1 could also play a role in mediating the effect of high K intake on K secretion.

Aldosterone and SGK

A large body of evidence suggests that SGK1 mediates, at least in part, the effect of aldosterone on renal K secretion [41, 88, 113, 116]. This notion is supported by studies performed in SGK1 knockout mice demonstrating that the phenotype of SGK1 deletion is similar to MR knockout mice and displays impaired renal K secretion in response to high dietary K intake [41]. The mechanism by which SGK1 stimulates renal K secretion includes enhancing the export of ROMK channels from the ER [140]. Recently, it has also been shown that WNK4 is the substrate of SGK1 which phosphorylates serine residue 1169 of its C terminus. Moreover, such SGK1-induced phosphorylation of WNK4 abolishes its inhibitory effect on ROMK channels in Xenopus oocytes [88]. However, the immunostaining study performed in SGK1 knockout mice shows that apical staining of ROMK channels in the CNT and CCD is normal or even intensified [41], suggesting that SGK1 is not essential for the export of ROMK channels. Thus, the stimulatory effect of aldosterone and SGK1 on K secretion may be mediated mainly by increasing Na transport. But it should be noted that in chronic experiments, aldosterone or SGK1 may play a permissive role in mediating the effect of high K intake on ROMK channels because high K intake failed to stimulate ROMK channels in adrenalectomized rats [81].

Role of AngII

Because high K intake suppresses the renin and AngII system [90, 102], it may play a role in modulating the effect of aldosterone or SGK1 on ROMK channels [46]. Figure 3 is a cell model illustrating the possible mechanism by which aldosterone-induced stimulation of SGK1 activates ROMK channels when the AngII system is down-regulated by high K intake. Thus, when AngII is suppressed, SGK1 stimulates the phosphorylation of WNK4 and thereby abolishes the WNK4-mediated inhibition of ROMK channels. In contrast, when the AngII signaling pathway is active under conditions of low Na intake, the stimulatory effect of SGK1 on ROMK channel is compromised.

Figure 4 is a scheme illustrating the role of the interaction between AngII and aldosterone/SGK1 in the regulating ROMK channels and K secretion during low Na intake. Unlike the condition of high K intake which suppresses renin–AngII pathway, low Na intake stimulates renin and AngII system. Because AngII has been shown to inhibit ROMK channels [130], the stimulatory effect of SGK1 on ROMK channels may be suppressed. Alternatively, AngII signaling pathway could directly modulate SGK1-WNK4 interaction and hence abolish the stimulatory effect of SGK1 on ROMK channels. This model could explain that infusion of aldosterone or low Na intake fails to stimulate ROMK channels and also that high K intake alone is not able to stimulate ROMK channels in the absence of aldosterone [81]. But the role of AngII in interacting with SGK1-WNK4 pathway is not explored and needs future experiments to prove the hypothesis.

Fig. 4.

A scheme showing the mechanism by which aldosterone and AngII regulate ROMK and BK channels in the CCD during low Na intake. The circle with x indicates the inhibition of a particular signaling pathway

Cytochrome P450 (CYP) epoxygenase

CYP epoxygenases such as CYP2C23 or CYP2J are expressed in the CNT and CCD [61, 71, 110]. Two lines of evidence suggest that CYP epoxygenase plays an important role in mediating the effect of high K intake on K secretion in the CCD [111]: (1) high K intake increased epoxyeicosatrienoic acid (EET) levels in the CCD and (2) 11,12-EET stimulates BK channel activity in the CCD. The expression of CYP2C23 is also increased in response to a high K diet [111]. The effect of high K on CYP2C23 is specific because high K intake does not increase CYP2J expression. The effect of high K intake on CYP2C23 expression is not due to high aldosterone level because low Na intake has been shown to decrease the expression of CYP2C23 [110]. Thus, CYP epoxygenase-dependent metabolism of arachidonic acid stimulates BK channels in response to a high K diet by an aldosterone-independent mechanism. This pathway may play a role in BK-dependent K secretion in the CCD and possibly in the CNT (Fig. 3).

Effect of low dietary K intake

K restriction decreases renal K secretion by inhibiting both apical ROMK and BK channels in PC [51, 127] as well as by stimulating K absorption [133, 135]. Figure 5 is a scheme of a cell model illustrating the current understanding regarding the effect of low K intake on apical K channels, Na channels, H,K-ATPase, and basolateral K channels in the CCD. Low K intake increases superoxide anion production which stimulates the expression of Src family PTK and the phosphorylation of p38 and ERK MAPKs [8]. Single-channel analysis in the CCD of mice and rats has demonstrated that the activity of both BK and ROMK-like SK decreases following activation of PTK and MAPKs [124, 129]. In addition, stimulation of ERK has been shown to inhibit epithelial Na channels (ENaC) [106] and accordingly diminish the driving force for K secretion. Moreover, K restriction stimulates K absorption [12, 28, 142] through enhancing colonic H,K-ATPase transcription [62, 103], and it is possible that PC may also be involved in K reabsorption [32]. K-restriction-induced increase in K reabsorption may also be the result of inhibiting apical K channels in IC [145, 148] because blocking K channels in IC prevents K recycling into the lumen and favors K absorption [145]. The molecular mechanism by which low K intake inhibits apical K channels in PC has been extensively studied. Figure 6 is a scheme illustrating the signaling pathway by which low K intake inhibits apical K channels in the CCD.

Fig. 5.

A cell scheme illustrating the mechanism by which low K intake inhibits K secretion in PC and stimulates K absorption in IC of the CCD. Solid arrow and dotted arrow indicate the enhanced and attenuated effect, respectively

Fig. 6.

A scheme showing the role of different signaling pathways in mediating the effect of low K intake on ROMK and BK channels in the CCD. The circle with x indicates the inhibition of a particular signaling pathway

Role of AngII

K restriction has been shown to stimulate renin and the AngII system [86, 90, 102]. Moreover, micropuncture studies have revealed that luminal perfusion of AngII inhibited K secretion in the distal nephron [118]. Patch-clamp experiments have further demonstrated that AngII down-regulates ROMK channels in the CCD and that such inhibition could be demonstrated only in the CCD from K-restricted rats [130]. Furthermore, the suppression of ROMK channels by AngII was absent by blocking NADPH oxidase or attenuated by inhibiting Src family PTK. This suggests that superoxide anions and PTK are involved in mediating the effect of angiotensin II.

MAPK

Low K intake has also been shown to activate MAPK such as p38 and ERK [7]. This effect is possibly mediated by superoxide anions because suppression of superoxide anions production abolished the effect of low K intake on MAPK activity [7,8]. The inhibitory role of p38 and ERK in the regulation of K secretion has been suggested by the observations that blocking p38 and ERK increases the ROMK channel activity and BK channel activity in the CCD [7, 51]. Moreover, the effect of MAPK on ROMK channels is independent of Src family PTK.

Src family PTK

Both ROMK and Src family PTK, c-Src, are expressed in the CCD [55]. ROMK channels are the substrate of Src family PTK, and it has been shown that tyrosine residue 337 of ROMK1 is a phosphorylation site of PTK. The tyrosine phosphorylation of ROMK is regulated by dietary K intake: A low K intake increases, whereas a high K intake decreases the level of the tyrosine-phosphorylated ROMK. Moreover, the level of Src family PTK such as c-Src and c-Yes increased in the renal cortex and outer medulla obtained from rats on a K-deficient diet and significantly decreased by high K intake [126]. Thus, it is possible that PTK is involved in mediating the inhibitory effect of low dietary K intake on the ROMK-like SK channels.

The role of PTK in mediating the effect of low K intake on K secretion is further supported by the finding that blocking PTK with herbimycin A significantly increased the activity of the ROMK-like SK channels in CCDs from rats on a KD diet [126]. However, the inhibitory effect of PTK on ROMK-like SK channels is not a direct consequence of the tyrosine phosphorylation because addition of exogenous c-Src does not inhibit the channel activity in excised patches [122]. Several lines of evidence indicate that stimulation of tyrosine phosphorylation of ROMK1 facilitates internalization of ROMK-like SK channels. First, inhibition of protein tyrosine phosphatase (PTP) decreases ROMK-like SK channel activity. This effect is absent in the presence of sucrose-containing bath or in CCDs treated with concanavalin A, an agent which inhibits endocytosis, [125], suggesting that inhibition of PTP increases the internalization of ROMK-like SK channels. Second, inhibition of PTP significantly increases tyrosine phosphorylation of ROMK1 and reduces the number of ROMK1 detected by confocal microscopic image and surface biotin labeling in HEK293 cells transfected with ROMK1 and c-Src [107]. Third, inhibition of PTP has no effect on K channel activity in cells transfected with the ROMK1 mutant, R1Y337A, indicating that phosphorylation of tyrosine residue 337 is essential for initiating the internalization of ROMK1 [107].

While stimulation of tyrosine phosphorylation enhances the internalization of ROMK1 channels, facilitating dephosphorylation has an opposite effect on ROMK1 channels. It has been demonstrated that stimulating tyrosine dephosphorylation increases the surface density of ROMK1 channels [67]. Moreover, the observation that inhibition of microtubule formation or application of tetanus toxin abolished the effect of herbimycin A on the ROMK channels in CCDs indicates that the effect of inhibiting PTK results from the stimulation of exocytosis [108, 128].

KS-WNK1 and WNK1

As discussed above, KS-WNK1 antagonizes effect on WNK1 which inhibits ROMK channels. K restriction has been reported to decrease the expression of KS-WNK1 and increase long form WNK1. Consequently, the antagonizing effect of KS-WNK1 on WNK1 is diminished, and WNK1-mediated inhibition of ROMK channels is enhanced in the CCD from animals fed on a low K diet [48].

Regulation of K transport in the CCD by hormones other than aldosterone and AngII

Vasopressin

Vasopressin plays an important role in stimulating renal K excretion during dehydration. A decrease in extracellular volume is expected to increase the plasma level of vasopressin which stimulates renal K excretion. The stimulatory effect of vasopressin on renal K excretion is partially due to increasing apical ROMK channel activity in the CCD by activation of V2 receptor and cAMP-dependent pathway [13]. Moreover, it has been shown that vasopressin increases K secretion in the distal tubule, including CNT and initial CCD, by stimulation of V1 receptor [3]. It has been reported that luminal vasopressin stimulates K secretion in distal tubule, and the effect of luminal vasopressin is abolished in the presence BK channel blocker, iberiotoxin. Thus, it is possible that stimulation of luminal V1 receptor activates BK channels by a Ca²⁺- and PKC-dependent mechanism [4].

PGE₂

Cyclooxygenase (COX) 1 and 2 are expressed in the CCD [139]. We have previously demonstrated that low K intake stimulates the COX2 expression and PGE₂ production in the rat kidney and that PGE₂ inhibits ROMK channels by a PKC-MAPK-dependent pathway [43], as shown in Fig. 6. Low K intake has been shown to stimulate renin production which increases PGE₂ production [86, 90, 102]. This mechanism may play a role in suppressing apical K channels during K restriction.

Uroguanylin and guanylin

Guanylin and uroguanylin have been shown to cause both hyperpolarization and depolarization of cultured CCD principal cells. The peptide-induced hyperpolarization and depolarization are blocked by protein kinase G and phospholipase A2, respectively [104, 105]. Micropuncture study has demonstrated that urogunylin stimulates BK-channel-dependent K secretion in rat distal nephron [5].

Prospect

Although our understanding regarding K transport in the CNT and CCD has been significantly extended recently, an integrated mechanism by which hormone and dietary K intake regulates K transport is still not completely understood. In the future study, it would be important to understand the connection and interaction among different kinases and pathways which regulate K secretion in the CCD. In addition, another focus would be to identify new molecular mechanisms and paradigm of renal K handling in the distal nephron. Recently, a study which measure simultaneously changes in plasma K and urinary K excretion demonstrates that increasing K loading with food intake in stomach stimulates renal K secretion even if plasma K concentration remains unchanged by K loading [49]. However, if K loading is not companied by stomach food feeding, raising plasma K concentration is observed before renal K secretion increases. This suggests a possible K sensing mechanism or gut factor present in the gastric tissue. Thus, it would be interesting to determine the nature of such a gut factor and the signaling mechanism by the gut factor regulates K transport in the collecting duct.