Distal tubule basolateral potassium channels: cellular and molecular mechanisms of regulation

Abstract

Purpose of review

Multiple clinical and translational evidence support benefits of high potassium diet; however, there many uncertainties underlying the molecular and cellular mechanisms determining effects of dietary potassium. K_ir4.1 and K_ir5.1 proteins form a functional heteromer (K_ir4.1/K_ir5.1), which is the primary inwardly rectifying potassium channel on the basolateral membrane of both distal convoluted tubule (DCT) and the collecting duct (CD) principal cells. The purpose of this mini-review is to summarize latest advances in our understanding of the evolution, physiological relevance and mechanisms controlling these channels.

Recent findings

K_ir4.1 and K_ir5.1 channels play a critical role in determining electrolyte homeostasis in the kidney and blood pressure, respectively. It was reported that K_ir4.1/K_ir5.1 serves as potassium sensors in the distal nephron responding to variations in dietary intake and hormonal stimuli. Global and kidney specific knockouts of either channel resulted in hypokalemia and severe cardiorenal phenotypes. Furthermore, knock out of K_ir5.1 in Dahl salt-sensitive rat background revealed the crucial role of the K_ir4.1/K_ir5.1 channel in salt-induced hypertension.

Summary

Here, we focus on reviewing novel experimental evidence of the physiological function, expression and hormonal regulation of renal basolateral inwardly rectifying potassium channels. Further investigation of molecular and cellular mechanisms controlling K_ir4.1 and K_ir4.1/K_ir5.1 mediating pathways and development of specific compounds targeting these channels function is essential for proper control of electrolyte homeostasis and blood pressure.

INTRODUCTION

Salt and water handling by the kidney directly impacts blood pressure. Ion channels and transporters in the distal tubules determine the transport rate and subsequently urinary excretion of electrolytes. The effect of sodium on blood pressure is dependent on diet composition, specifically on the sodium/potassium ratio. Diets supplemented with high potassium are associated with a lower risk of major cardiovascular events [1,2]. Despite highly relevant clinical and translational evidence supporting benefits of high potassium diet, there is a substantial lag in our understanding of the underlying molecular mechanisms. Inwardly rectifying K⁺ channels, specifically K_ir4.1 and K_ir4.1/K_ir5.1 (encoded by Kcnj10 and Kcnj16 genes, respectively), play a dominant role in determining blood potassium level and serve as potassium sensors in the distal nephron [3–5]. This brief review discusses recent advances in our understanding of potassium homeostasis mediated by the basolateral K⁺ channels in the distal nephron and mechanisms controlling the activity of these channels.

THE EVOLUTION OF K⁺ CHANNELS

Potassium channels expressed in virtually all type of eukaryotic cells and involved in a plethora of physiological processes in both electrically excitable and non-excitable cells. The evolution and diversification of ion channels often provide important insights in the understanding of their function. From the structural characteristics and genome sequencing data majority of K⁺ channels has been traced back to the prokaryotic world [6]. Potassium channels could be divided into three groups based on the principal structural components – two transmembrane helices inwardly rectifier (K_ir), two pore four transmembrane (K_2p) and six membrane helix voltage gated outward rectifier (K_v) (Figure 1). In the vertebrate, members of the K_ir family are further divided into the three main evolutional groups – elementary, ATP-regulated (exhibit rundown in the absence of cytoplasmic ATP as a result of the net de-phosphorylation of the channel [7]), and G-protein-activated channels [8]. The last two evolutional groups of K_ir genes are the major classes of the K_ir family, and their expression must be mutually exclusive according to phylogenetic analyses [9]. Indeed, G-protein-activated channels are expressed in the neuronal cells, and ATP-regulated K_ir channels are represented in the epithelial and glial tissues [10,11]. ATP-sensitive (K_ATP) channels composed of K_ir and sulfonylurea receptor (SUR) subunits set aside and not reviewed here. The ATP-regulated K_ir4.1 (encoded by Kcnj10 gene), K_ir1.1 (renal outer medullary potassium channel (ROMK); encoded by Kcnj1) and K_ir7.1 (Kcnj13), and elementary K_ir5.1 (Kcnj16) channels are highly expressed in the distal kidney tubules. ROMK channels are expressed on the apical plasma membrane [12], and K_ir7.1 mRNA was found in the intercalated cells of cortical collecting duct (CCD) [13]. This brief review is focused on the mechanisms controlling expression and activity of the basolateral K_ir4.1 and K_ir4.1/K_ir5.1 channels expressed in distal convoluted tubule (DCT) and the collecting duct (CD) principal cells. It should be noted that these channels are highly expressed in CNS and contribute to a number of neurological disorders such as depression, epileptic seizure and others [14–18].

FIGURE 1.

A schematic of phylogenetic groups of K⁺ channels.

BASOLATERAL CHANNEL CONDUCTANCE AND HETEROMERIZATION IN THE DISTAL TUBULES

ATP-regulated K_ir, such as apical ROMK and basolateral K_ir4.1 channels, are considered as “weak” inwardly rectifiers. Namely, “weak” indicates a notable persistent outward current when compared with “strong” rectifiers, where outward conductance is virtually abolished due to intracellular factors. K_ir4.1 can function as a homomeric channel or may form a heteromeric channel with K_ir5.1 [19]. K_ir4.1/K_ir5.1 heteromeric channel exhibit biophysical properties distinct from those of homomeric K_ir4.1 channel. This includes a larger single channel conductance and greater pH sensitivity [20,21]. It is important to note that K_ir4.1/K_ir5.1 is the predominant K⁺ basolateral channel and a key determinant of the resting membrane potential in both DCT and CD principal cells [22,23]. This notion is supported by both electrophysiological and pharmacological methods. Thus, it was shown that K_ir4.1 homomeric channels are highly expressed in brain astrocytes and can be blocked with fluoxetine [24,25]. In contrast, fluoxetine has no effect on the macroscopic K⁺-selective conductance in CCD principal cells, which could be abolished by the tricyclic antidepressant nortriptyline [26] (nortriptyline inhibits both homomeric K_ir4.1 and heteromeric K_ir4.1/K_ir5.1 channels). Interestingly, the most common cardiovascular complication of nortriptyline, which is FDA-approved drug for the treatment of major depression, is hypotension [27,28].

A number of human mutations in KCNJ10, which underlie SeSAME/EAST syndrome, are characterized by salt wasting, renal tubulopathy, and autosomal recessive epilepsy, ataxia, sensorineural deafness [29,30]. Mutations in KCNJ16 gene were reported to cause non-familial Brugada syndrome associated with sudden cardiac death [31]. Deletion of K_ir5.1 in mice strongly decrease sensitivity to intracellular pH and results in elevated open channel probability of compensatory overexpressed homomeric K_ir4.1 channel in the distal nephron [32]. Furthermore, it was recently reported that mutations in the hepatocyte nuclear factor 1 homeobox B (HNF1β) cause autosomal dominant tubulointerstitial kidney disease [33]. As was shown in this study, compromised K_ir5.1, and potentially K_ir4.1 and the thiazide-sensitive sodium-chloride cotransporter (NCC; encoded by SLC12A3), drive these effects [33]. However, it should be noted that K_ir5.1 is less studied compared to K_ir4.1 and no human mutations in KCNJ16 mimicking SeSAME/EAST syndrome have been identified up-to-date. One of the potential reasons for this is that in contrast to KCNJ10, KCNJ16 gene is not included in the database of genes for the screening. Importantly, variations of the gene encoding the K_ir4.1 subunit results in a loss of function of both homomeric K_ir4.1 and heteromeric K_ir4.1/K_ir5.1 channels [34]. Furthermore, we recently reported that functional K⁺ conductance mediated by basolateral K_ir channels requires K_ir5.1 subunit for trafficking of K_ir4.1 subunit towards the plasma membrane and compensatory overexpression of homomeric K_ir4.1 observed in K_ir5.1 knockout rats did not restore electrolyte balance [35]. Taken together these results indicate that both K_ir4.1 and K_ir5.1 are essential for proper inward rectification and basolateral potassium conductance along the distal nephron.

DIETARY EFFECTS

The thiazide-sensitive NCC is sodium/chloride transporter localized to the apical membrane of the whole DCT, and ENaC channel, which expression begins at the late segments of DCT (DCT2; where both NCC and ENaC are present) and continues throughout the rest of the aldosterone-sensitive distal nephron, are the major contributors to sodium reabsorption at these sites and are involved in the final step of the blood pressure regulation in the kidney [36,37]. Dietary potassium intake has antihypertensive effects and improves cardiovascular outcomes [2,38]. Recent studies provide insights into the effect of high K⁺ diet on the basolateral K_ir4.1 and K_ir4.1/K_ir5.1 channels in both DCT and CCD [39,40]. It was reported that high K⁺ chow (5%; exact diet composition and chloride level were not disclosed) inhibited, and conversely, K⁺ deficient chow (<0.001%) activated the K_ir4.1/K_ir5.1 heteromer in the DCT [40]. Furthermore, it was shown that high K⁺ intake promoted an inhibition, whereas low K⁺ intake increased the abundance of the expression in NCC and sodium transport in DCT. In contrast, dietary potassium did not have any effects on NCC expression in kidney-specific K_ir4.1 knockout mice [40]. Opposed to the inhibitory effects of high consumption on K_ir4.1/K_ir5.1 channels in DCT, treatment of mice with high K⁺ chow (6% K⁺ and either high (5%) or regular Cl^– (0.5%)) elicited an increase of K_ir4.1/K_ir5.1 channels activity as well as subsequent Na⁺ transport through ENaC in the CCD principal cells (Table 1). Furthermore, stimulation of aldosterone signaling by Deoxycorticosterone acetate (DOCA) recapitulated the effects of high K⁺ diet on K_ir4.1/K_ir5.1 channels in CCD [39]. The same studies also provide evidence that the increase in sodium content (from 0.3% to 1.6% NaCl) resulted in the reduction in basolateral K⁺-selective conductance in CCD likely reflecting reduced circulating aldosterone levels. Interestingly, earlier studies by Lachleb et al. showed that dietary potassium intake (either 8% K⁺, 0.3% Na⁺ or 0% K⁺, 0.3% Na⁺) had no influence on the properties of K_ir4.1/K_ir5.1 channels, when compared to the regular diet (0.6 K⁺, 0.3% Na⁺), although there was a notable tendency of a higher K_ir4.1/K_ir5.1 activity on high K⁺ compared to the low K⁺ state. Furthermore, a Na⁺-depleted diet (0.09% Na⁺, 0.6% K⁺) moderately increased its open probability [41].

Table 1.

Scheme of regulation of K_ir4.1/K_ir5.1 channels in DCT and CCD by high K⁺ diet based on recent studies by Wang et al. [40] and Tomilin et al. [39].

High K+ diet
DCT	CCD
Kir4.1/Kir5.1 ↓	Kir4.1/Kir5.1 ↑
NCC ↓ ENaC ?	ENaC ↑
ROMK ↑	ROMK ↑
CLC-K2/b ↓	CLC-K2/b ↓

MOLECULAR AND CELLULAR SIGNALING MECHANISMS CONTROLLING K_ir4.1/K_ir5.1 CHANNELS

Multiple factors regulate basolateral potassium channels coupled with the apical transport process in distal kidney tubules to regulate K⁺ homeostasis in the body. Imbalance in basolateral potassium sensing promote strong changes in serum concentrations and often leading to hypokalemia, or hyperkalemia. Generally, basolateral K⁺ conductance is higher than the apical one and possibly plays a dominant role in the control of potassium balance [42]. Functionally, K_ir4.1/K_ir5.1 channel recycles K⁺ ions across the basolateral membrane, moved into the cell by the Na⁺/K⁺-ATPase, and thus setting a negative basolateral membrane potential to drive Na⁺ reabsorption and control K⁺ secretion in DCT and CCD [3,4]. Using the specific deletion of K_ir4.1 gene in the kidney tubules (part of the thick ascending limb (TAL), DCT, the connecting tubule (CNT) and CCD) Cuevas et al. shows that K_ir4.1/K_ir5.1 channel is required to sense and control plasma K⁺ as well as modulate NCC-mediated NaCl transport in DCT [43]. Similarly, to K_ir4.1 knockout, the deletion of K_ir5.1 in mice promotes hypokalemia, associated with increased renal K⁺ excretion [32].

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is considered as major specific physiological activator of almost all K_ir channels since these channels have several conserved PIP₂-binding sites [44]. Interestingly, the deletion of 6 amino acids in the transmembrane domain 2 of K_ir5.1, which is localized within the region of the PIP₂ binding site, resulted in a complete knock out of this protein and a striking severe cardiorenal phenotype [35]. It was also reported that dopamine, that can induce stimulation of PIP₂ hydrolysis and subsequent activation of PKC [45], inhibits K_ir4.1/K_ir5.1 channels activity through the D2-like receptors [46]. PIP₂ and other phosphatidylinositides are essential regulators of renal ion channels [47–50], and additional studies are required to address potential modulation of K_ir4.1 and K_ir4.1/K_ir5.1 channels by phosphatidylinositides. Opposite to dopamine inhibitory effect, insulin and, to a lesser extent, IGF-1 can activate K_ir4.1/K_ir5.1 channel activity and increase open probability triggering hyperpolarization of the basolateral membrane and facilitating sodium reabsorption [26].

Angiotensin II (Ang II) and aldosterone, main components of the renin–angiotensin–aldosterone system are well known factors mediating apical K⁺ channel excretion [51,52]. Both Ang II and aldosterone, regulate through corresponding receptors (angiotensin (AT₁ and AT₂) and mineralocorticoid (MR) receptors, respectively) extracellular fluid volume and plasma K⁺ levels by controlling ion channels and transporters in the distal nephron. The plasma K⁺ concentration is recognized as an important regulator of renal ion transport mechanisms, and it is believed that K_ir4.1/K_ir5.1 channels play important role in sensing the changes in plasma K⁺ levels [43,53–56]. Importantly, changes in plasma K⁺ levels can modulate sodium absorption, including NCC-mediated transport in DCT, independently of aldosterone. Wu et al. recently attempted to delineate a possible signaling pathway which mediates the effect of high K⁺ diet on K_ir4.1/K_ir5.1 and NCC activity in DCT and tested contribution of Ang II-mediated pathway. While Ang II failed to affect channel activity at the baseline, the hormone inhibited K_ir4.1/K_ir5.1 in the presence of AT₁R blocker, losartan, suggesting that AT₂R is responsible for mediating the inhibitory effect of Ang II on basolateral K⁺ channels in the DCT [57]. These data are consistent with previously reported findings from the same group that increased dietary K⁺ intake inhibits the K_ir4.1/K_ir5.1 channels [40]. However, as discussed in the accompanying editorial commentary by Patel and Hussain [58], there seems to be a disconnect between the acute patch-clamp experiment and the 4-day in vivo renal clearance studies.

K_ir4.1/K_ir5.1 REGULATION OF BLOOD PRESSURE

The expression of K_ir4.1/K_ir5.1 channels in kidney tubules directly correlates with all main sodium pathways and functionally bound to sodium transport mediated by NKCC2 (in TAL), NCC (DCT) and ENaC (DCT2 and CCD). The full blockade or knockout of renal K_ir4.1/K_ir5.1 channels depolarize basolateral membrane in all described nephron segments and could be considered as equivalent to antihypertensive treatment by three types of diuretics (loop (TAL; furosemide), thiazide (NCC; hydrochlorothiazide) and potassium-sparing (CCD; amiloride)) at the same time. Recent studies using genetically modified animal research models reveal relation of these channels to the blood pressure regulation. On the genetic level, silencing of K_ir4.1 gene strongly associated with neurological pathologies, like ataxia and other, as well as early mortality. In contrast, the mutation in K_ir5.1 did not result in mortality or any visible neurological pathologies present in K_ir4.1 phenotype [32,35]. To demonstrate the functional importance of K_ir4.1/K_ir5.1 in blood pressure regulation and potential treatment of hypertension, we created the knockout of K_ir5.1 in Dahl salt-sensitive (SS) rats [35]. The deletion of K_ir5.1 channel promotes not only the attenuation in basal mean arterial pressure in Dahl SS rats but completely prevent from the development of salt-induced hypertension and renal damage following high salt diet when animal’s diet was supplemented with high potassium. High potassium nutritional content is necessary for K_ir5.1 knockout survival, since the deletion of the K_ir4.1/K_ir5.1 channel triggered salt wasting phenotype resulted in hypokalemia, similarly to the phenotype observed in K_ir4.1 knockout, which further in combination with elevated diuresis triggered rapid mortality during a high salt challenge. Overall, these results indicate that the combination of two key factors, high potassium diet and control of renal basolateral conductance or K_ir4.1/K_ir5.1 channel activity, have enormous potential as a means to reduce blood pressure and control hypertension. The broad significance of this study is the identification of the indispensable importance of K_ir4.1/K_ir5.1channels in the development and pathology of salt-sensitive hypertension.

CONCLUSION

The blood pressure associated with the renal regulation of fluid and electrolyte balance. As was discussed above, remodeling of inward rectifier K⁺ channels function and particularly basolateral K_ir4.1/K_ir5.1 has the potential for tuning chronic blood pressure and, thus, developing new pharmacological tools for treatment of hypertension. Renal basolateral K_ir4.1/K_ir5.1 channels represent potential targets for therapeutic intervention in the fight against serum potassium disturbances. Despite all recent knowledge in our understanding of the importance of the basolateral K_ir4.1/K_ir5.1 channel for K⁺ homeostasis, the molecular mechanisms and complete scheme of the regulation of this channel function along the distal nephron in health and disease conditions requires future extensive attention.

KEY POINTS.

• Renal basolateral K_ir channels are critical in the control of electrolyte homeostasis in the kidney and blood pressure

• K_ir4.1 and K_ir5.1 (encoded by Kcnj10 and Kcnj16, respectively) form heteromeric channel (K_ir4.1/K_ir5.1) in the distal convoluted tubules and principal cells of collecting duct

• K_ir4.1 and K_ir4.1/K_ir5.1 are regulated by dietary potassium content as well as various hormonal and metabolic factors