Abstract-Primary hyperaldosteronism, one cause of which is aldosterone-producing adenomas (APAs), may account for Յ5% to 10% of cases of essential hypertension. Germline mutations have been identified in 2 rare familial forms of primary hyperaldosteronism, but it has been reported recently that somatic mutations of the KCNJ5 gene, which encodes a potassium channel, are present in some sporadic nonsyndromic APAs. To address this further we screened 2 large collections of sporadic APAs from the United Kingdom and Australia (totalling 73) and found somatic mutations in the selectivity filter of KCNJ5 in 41% (95% CI: 31% to 53%) of the APAs (30 of 73). These included the previously noted nonsynonymous mutations, G151R and L158R, and an unreported 3-base deletion, delI157, in the region of the selectivity filter. APAs containing a somatic KCNJ5 mutation were significantly larger than those without ( Key Words: hyperaldosteronism Ⅲ hypertension Ⅲ potassium channels Ⅲ KCNJ5 Ⅲ aldosterone-producing adenoma Ⅲ posture response P rimary hyperaldosteronism (PA) is now recognized as a common, treatable, and potentially curable form of hypertension, which may account for Յ10% of cases of socalled essential hypertension. [1][2][3] Most cases of PA are sporadic and result from 2 major types of adrenal pathology, an aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia. In recently published series, the frequency of APA varied between 28% and 50% of patients with PA. 4 Choi et al 5 recently reported somatic mutations in a potassium channel, KCNJ5 (also called GIRK4 or Kir3.4), in 8 of 20 APAs studied and a germline mutation in the same gene in all 3 affected members of a family with florid, early onset, nondexamethasone-suppressible PA associated with marked hyperplasia of zona fasciculata (ZF), suggesting a novel pathway that might activate growth of aldosteronesecreting cells. These mutations within the selectivity filter of the potassium channel reduce the normal K ϩ /Na ϩ selectivity of the channel, and the resulting depolarization of the adrenocortical cell could lead to calcium loading and growth. However, the APAs carrying KCNJ5 mutations were large (mean of 2.8 cm and all Ͼ2 cm in diameter) and might represent a subgroup with a phenotype more relevant to the giant hyperplastic adrenals seen in the family with the germline KCNJ5 mutation. 5 To address this issue we have screened a large collection of APAs (totalling 73) from geographically distinct centers (United Kingdom and Australia) to determine whether somatic mutations of KCNJ5 are present in unselected APAs regardless of size. We also

P rimary hyperaldosteronism (PA) is now recognized as a common, treatable, and potentially curable form of hypertension. In fact it may account for ≥10% of cases of what would previously have been labeled as essential hypertension.1,2 The excessive aldosterone production usually derives from either an aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia. The balance between these 2 pathologies varies; however, in most recently published series, bilateral adrenal hyperplasia is about twice as common as APA. 3,4 Although the majority of PA is sporadic, there are monogenic familial forms of the condition (familial hyperaldosteronism types I, II, and III [FH-I, FH-II, and FH-III]). The molecular basis for FHI, glucocorticoid-remediable aldosteronism, has been well understood for >2 decades and involves a recombination event that places the aldosterone synthase enzyme, CYP11B2, under the control of ACTH. 5,6 In contrast, the molecular genetics for FHIII has been resolved recently with the discovery that they are caused by germline mutations that cluster in the selectivity filter of a potassium channel, KCNJ5. 7-9 These mutations affect the Na + permeability of the channel, which is thought to lead to depolarization of zona glomerulosa cells in the adrenal cortex thereby activating aldosterone release.Before the discovery of selectivity filter mutations in KCNJ5, this potassium channel was not even recognized as being expressed in the human adrenal gland. However, it is now known to be important in sporadic as well as the much rarer syndromic forms of PA, 10 with ≤40% of APAs carrying somatic mutations in the same KCNJ5 gene. 7,11 The coding sequence for KCNJ5 has been resequenced in a large collection of adrenal lesions (both APAs and non-APAs) without identifying further somatic mutations outside of the selectivity filter itself. 12However, we hypothesized that rare variants, in the form of See Editorial Commentary, pp 668-669Abstract-Primary aldosteronism (autonomous aldosterone production with suppressed renin) plays an important pathophysiological role in what has been previously labeled as essential hypertension. Besides the recently described germline mutations in the KCNJ5 potassium channel associated with familial primary aldosteronism, somatic mutations in the same channel have been identified within aldosterone-producing adenomas. In this study, we have resequenced the flanking and coding region of KCNJ5 in peripheral blood DNA from 251 white subjects with primary aldosteronism to look for rare variants that might be important for the pathophysiology of sporadic primary aldosteronism. We have identified 3 heterozygous missense mutations (R52H, E246K, and G247R) in the cohort and found that 12 (5% of the cohort) were carriers for the rare nonsynonymous single nucleotide polymorphism rs7102584 causing E282Q substitution of KCNJ5. By expressing the channels in Xenopus oocytes and human adrenal H295R cells, we have shown that the R52H, E246K, and E282Q substitutions are functional, but the G247R...

The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect Na+ fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the NH2 terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK.

Abstract-We identified a new kindred with the familial syndrome of hypertension and hyperkalemia (pseudohypoaldosteronism type II or Gordon's syndrome) containing an affected father and son. Mutation analysis confirmed a single heterozygous G to C substitution within exon 7 (1690GϾC) that causes a missense mutation within the acidic motif of WNK4 (564DϾH). We confirmed the function of this novel mutation by coexpressing it in Xenopus oocytes with either the NaCl cotransporter (NCCT) or the inwardly rectifying K-channel (ROMK). Wild-type WNK4 inhibits 22 Na ϩ flux in Xenopus oocytes expressing NCCT by Ϸ90% (PϽ0.001), whereas the 564DϾH mutant had no significantly inhibitory effect on flux through NCCT. In oocytes expressing ROMK, wild-type WNK4 produced Ͼ50% inhibition of steady-state current through ROMK at a ϩ20-mV holding potential (PϽ0.001). The 564DϾH mutant produced further inhibition with steady-state currents to some 60% to 70% of those seen with the wild-type WNK4. Using fluorescent-tagged NCCT (enhanced cyan fluorescent protein-NCCT) and ROMK (enhanced green fluorescent protein-ROMK) to quantify the expression of the proteins in the oocyte membrane, it appears that the functional effects of the 564DϾH mutation can be explained by alteration in the surface expression of NCCT and ROMK. Compared with wild-type WNK4, WNK4 564DϾH causes increased cell surface expression of NCCT but reduced expression of ROMK. This work confirms that the novel missense mutation in WNK4, 564DϾH, is functionally active and highlights further how switching charge on a single residue in the acid motif of WNK4 affects its interaction with the thiazide-sensitive target NCCT and the potassium channel ROMK.

The WNK (with no lysine kinase) kinases are a novel class of serine/threonine kinases that lack a characteristic lysine residue for ATP docking. Both WNK1 and WNK4 are expressed in the mammalian kidney, and mutations in either can cause the rare familial syndrome of hypertension and hyperkalemia (Gordon syndrome, or pseudohypoaldosteronism type 2). The molecular basis for the action of WNK4 is through alteration in the membrane expression of the NaCl co-transporter (NCCT) and the renal outer-medullary K channel KCNJ1 (ROMK). The actions of WNK1 are less well defined, and evidence to date suggests that it can affect NCCT expression but only in the presence of WNK4. The results of co-expressing WNK1 with ROMK in Xenopus oocytes are reported for the first time. These studies show that WNK1 is able to suppress total current directly through ROMK by causing a marked reduction in its surface expression. The effect is mimicked by a kinase-dead mutant of WNK1 (368D>A), suggesting that it is not dependent on its catalytic activity. Study of the time course of ROMK expression further suggests that WNK1 accelerates trafficking of ROMK from the membrane, and this effect seems to be dynamin dependent. Using fragments of full-length WNK1, it also is shown that the effect depends on residues in the middle section of the protein (502 to 1100 WNK1) that contains the acidic motif. Together, these findings emphasize that the molecular mechanisms that underpin WNK1 regulation of ROMK expression are distinct from those that affect NCCT expression.J Am Soc Nephrol 17: 1867 -1874, 2006 . doi: 10.1681 T he WNK (with no lysine kinase) kinases WNK1 and WNK4 are widely expressed in mammalian transporting epithelia (1,2), and expression studies in Xenopus oocytes suggest that they are able to modify the expression of several co-transporters and ion channels (3,4). The details of the interaction are best understood for WNK4, which reduces surface expression of the thiazide-sensitive NaCl co-transporter (NCCT; gene symbol SLC12A3) in Xenopus oocytes (5-8). This effect of WNK4 depends on its serine-threonine (S/T) kinase activity as well as a highly conserved downstream acidic motif (EPEEPEADQH). Mutations that cause charge-changing amino acid substitutions within this motif abolish the inhibitory effect of wild-type WNK4 and cause the phenotype of hypertension and hyperkalemia that characterizes Gordon syndrome (pseudohypoaldosteronism type 2 [PHA2]; OMIM #145260) (9). WNK1 mutations also can cause this phenotype, but published data suggest that WNK1 protein is effective only in regulating NCCT trafficking when coexpressed with WNK4 (5,10). This suggests that the WNK may form a multimeric complex with NCCT and that protein-protein interactions between WNK1 and WNK4 are key to the functionality of WNK1.It is not known whether this paradigm of WNK1-WNK4 interaction extends to the effects of WNK on other transporters or ion channels. Lifton's laboratory has shown, for example, that WNK4 also inhibits expression of the Na-K-Cl cotransporter SLC1...

Hypertension (high blood pressure) is a major public health problem affecting more than a billion people worldwide with complications, including stroke, heart failure and kidney failure. The regulation of blood pressure is multifactorial reflecting genetic susceptibility, in utero environment and external factors such as obesity and salt intake. In keeping with Arthur Guyton's hypothesis, the kidney plays a key role in blood pressure control and data from clinical studies; physiology and genetics have shown that hypertension is driven a failure of the kidney to excrete excess salt at normal levels of blood pressure. There is a number of rare Mendelian blood pressure syndromes, which have shed light on the molecular mechanisms involved in dysregulated ion transport in the distal kidney. One in particular is Familial hyperkalemic hypertension (FHHt), an autosomal dominant monogenic form of hypertension characterised by high blood pressure, hyperkalemia, hyperchloremic metabolic acidosis, and hypercalciuria. The clinical signs of FHHt are treated by low doses of thiazide diuretic, and it mirrors Gitelman syndrome which features the inverse phenotype of hypotension, hypokalemic metabolic alkalosis, and hypocalciuria. Gitelman syndrome is caused by loss of function mutations in the thiazide-sensitive Na/Cl cotransporter (NCC); however, FHHt patients do not have mutations in the SCL12A3 locus encoding NCC. Instead, mutations have been identified in genes that have revealed a key signalling pathway that regulates NCC and several other key transporters and ion channels in the kidney that are critical for BP regulation. This is the WNK kinase signalling pathway that is the subject of this review.

This novel KCNJ5 mutation behaves like the three selectivity filter mutations previously reported in APAs depolarizing the cell and showing reduced cation selectivity. The reduced sensitivity to tertiapin-Q suggests that the abnormal Na(+) permeability of these selectivity mutations does indeed reflect structural changes around the mouth of the ion channel.

1 Calu-3 cells have been used to investigate the actions of 4-chloro-benzo [F]isoquinoline (CBIQ) on short-circuit current (SCC) in monolayers, whole-cell recording from single cells and by patch clamping. 2 CBIQ caused a sustained, reversible and repeatable increase in SCC in Calu-3 monolayers with an EC 50 of 4.0 mM. Simultaneous measurements of SCC and isotopic fluxes of 36 Cl À showed that CBIQ caused electrogenic chloride secretion. 3 Apical membrane permeabilisation to allow recording of basolateral membrane conductance in the presence of a K þ gradient suggested that CBIQ activated the intermediate-conductance calcium-sensitive K þ -channel (KCNN4). Permeabilisation of the basolateral membranes of epithelial monolayers in the presence of a Cl À gradient suggested that CBIQ activated the Cl À -channel CFTR in the apical membrane. 4 Whole-cell recording in the absence of ATP/GTP of Calu-3 cells showed that CBIQ generated an inwardly rectifying current sensitive to clotrimazole. In the presence of the nucleotides, a more complex I/V relation was found that was partially sensitive to glibenclamide. The data are consistent with the presence of both KCNN4 and CFTR in Calu-3. 5 Isolated inside-out patches from Calu-3 cells revealed clotrimazole-sensitive channels with a conductance of 12 pS at positive potentials after activation with CBIQ and demonstrating inwardly rectifying properties, consistent with the known properties of KCNN4. Cell-attached patches showed single channel events with a conductance of 7 pS and a linear I/V relation that were further activated by CBIQ by an increase in open state probability, consistent with known properties of CFTR. It is concluded that CBIQ activates CFTR and KCNN4 ion channels in Calu-3 cells.