Meena Murthy

Somatic Mutations Affecting the Selectivity Filter of KCNJ5 Are Frequent in 2 Large Unselected Collections of Adrenal Aldosteronomas

Primary hyperaldosteronism, one cause of which is aldosterone-producing adenomas (APAs), may account for ⩽5% 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 (1.61 cm [95% CI: 1.39–1.83 cm] versus 1.04 cm [95% CI: 0.91–1.17 cm]; P<0.0001) but with substantial overlap in size between genotypes. The APAs carrying a mutation, but not those without, also consistently lacked a postural aldosterone response, suggesting a physiologically distinct subtype. Hence, somatic KCNJ5 mutations are not restricted to large APAs (>2 cm), and their frequency in our unselected series suggests they are common and could be important in the molecular pathogenesis of many sporadic cases of APA.

WNK1 affects surface expression of the ROMK potassium channel independent of WNK4.

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.

A new kindred with pseudohypoaldosteronism type II and a novel mutation (564D>H)in the acidic motif of the WNK4 gene.

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 (1690G>C) that causes a missense mutation within the acidic motif of WNK4 (564D>H). 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 22Na+ flux in Xenopus oocytes expressing NCCT by ≈90% (P<0.001), whereas the 564D>H mutant had no significantly inhibitory effect on flux through NCCT. In oocytes expressing ROMK, wild-type WNK4 produced >50% inhibition of steady-state current through ROMK at a +20-mV holding potential (P<0.001). The 564D>H 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 564D>H mutation can be explained by alteration in the surface expression of NCCT and ROMK. Compared with wild-type WNK4, WNK4 564D>H causes increased cell surface expression of NCCT but reduced expression of ROMK. This work confirms that the novel missense mutation in WNK4, 564D>H, 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.

Role for Germline Mutations and a Rare Coding Single Nucleotide Polymorphism Within the KCNJ5 Potassium Channel in a Large Cohort of Sporadic Cases of Primary Aldosteronism

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 mutation is indistinguishable from wild type. Although the functional substitutions are remote from the selectivity filter, they affect the inward-rectification, the ability of the KCNJ5 channels to conduct Na+ currents and ATII-induced aldosterone release from the H295R cell line. Together these data suggest that germline variation in the KCNJ5 gene has a role to play in the common sporadic form as well as the much rarer syndromic forms of primary aldosteronism.

Characterization of a novel somatic KCNJ5 mutation delI157 in an aldosterone-producing adenoma.

Objective: Adrenal aldosterone-producing adenomas (APAs) are an increasingly recognized cause of primary aldosteronism, and somatic mutations within the KCNJ5 gene encoding an inwardly rectifying K+ channel (also called GIRK4 or Kir3.4) have been identified by several groups including our own. We identified the previously noted G151R and L168R mutations in the region of a selectivity filter of the channel as well as a previously unreported 3-base deletion, delI157. Here, we report the functional properties of KCNJ5 channels carrying this novel delI157 mutation. Methods: The delI157 mutation was introduced into wild-type KCNJ5 sequences to allow its expression in both H295R cells and Xenopus oocytes to study its expression and electrophysiology, respectively. Results: In the adrenal cell line H295R, the delI157 mutant expresses and traffics normally to the cell surface. However, the current–voltage behavior of the mutant in oocytes is distinct from wild-type channels and mimics closely other selectivity filter mutations. In particular, its ability to support substantial current when extracellular K+ is replaced by Na+. We also report for the first time that the mutants have reduced sensitivity to the KCNJ5 inhibitor tertiapin-Q that binds to the external vestibule of the channel pore. Conclusion: 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.

4‐Chloro‐benzo[F]isoquinoline (CBIQ) activates CFTR chloride channels and KCNN4 potassium channels in Calu‐3 human airway epithelial cells

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. CBIQ caused a sustained, reversible and repeatable increase in SCC in Calu‐3 monolayers with an EC50 of 4.0 μM. Simultaneous measurements of SCC and isotopic fluxes of 36Cl− showed that CBIQ caused electrogenic chloride secretion. Apical membrane permeabilisation to allow recording of basolateral membrane conductance in the presence of a K+ gradient suggested that CBIQ activated the intermediate‐conductance calcium‐sensitive K+‐channel (KCNN4). Permeabilisation of the basolateral membranes of epithelial monolayers in the presence of a Cl− gradient suggested that CBIQ activated the Cl−‐channel CFTR in the apical membrane. 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. 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.

The acidic motif of WNK4 is crucial for its interaction with the K channel ROMK

WNK kinases have rapidly emerged as important regulators of Na+ and K+ homoeostasis in the mammalian kidney where they regulate the trafficking of proteins such as the NaCl-cotransporter (NCCT) and K+ channel, ROMK. However, an increasing number of WNK effects are kinase-independent, including their interaction with ROMK, and involve instead protein-protein interactions. Outside of their kinase domain all WNKs contain a unique run of predominantly negatively charged amino acids dubbed the acidic motif, where the WNK4 disease mutations causing Gordons syndrome also cluster. To look further at the role of this motif we studied the effects of WNK4 fragments, including one with a deleted acidic motif (DeltaAM) and a 10-mer acidic motif peptide on ROMK expression in Xenopus oocytes. We found that an N-terminal fragment of WNK4 (1-620 WNK4) containing the acidic motif retains full activity in inhibiting ROMK currents. However, DeltaAM WNK4 is completely inactive and the effect of WNK4 or 1-620 WNK4 can be completely blocked by co-injection of the 10-mer acidic motif peptide. The blocking action of the peptide was sequence specific as a peptide with a randomised sequence was inactive. These results on ROMK currents were paralleled by changes in membrane expression of fluorescent EGFP-ROMK. Finally, we show that 1-620 WNK4 can pull down ROMK and this interaction can be blocked with the acidic motif peptide. These results confirm the important role of the acidic motif of WNK4 in its protein-protein interaction with the ROMK channel.

WNK signalling pathways in blood pressure regulation

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.

Neural control of submucosal gland and apical membrane secretions in airways

The mechanisms that lay behind the low‐level secretions from airway submucosal glands and the surface epithelium in the absence of external innervation have been investigated in small areas (1.0–1.5 cm2) of mucosa from sheep tracheas, freshly collected from a local abattoir. Glandular secretion was measured by an optical method while short circuit current was used as a measure of surface secretion. Activation of neurones in the intrinsic nerve net by veratrine alkaloids caused an immediate increase in both glandular secretion and short circuit current, both effects being blocked by the addition of tetrodotoxin. However, agents known to be acting directly on the glands, such as muscarinic agonists (e.g., carbachol) or adenylate cyclase activators (e.g., forskolin) were not influenced by tetrodotoxin. The toxin alone had no discernable effect on the low‐level basal secretion shown by unstimulated glands. Calu‐3 cell monolayers, generally agreed to be a surrogate for the secretory cells of submucosal glands, showed no sensitivity to veratrine alkaloids, strengthening the view that the veratrine‐like drugs acted exclusively on the intrinsic nerve net. The data are discussed in relation way in which transplanted lungs can maintain mucociliary clearance and hence a sterile environment in the absence of external innervation, as in transplanted lungs.

9B.03: A NOVEL INSERTIONAL SOMATIC KCNJ5 MUTATION IN AN AUSTRALIAN PATIENT WITH AN ALDOSTERONE PRODUCING ADENOMA.

Objective: Primary aldosteronism (PA), in which there is excessive and autonomous adrenal production of aldosterone, accounts for around 5–10% of hypertension. PA may be unilateral (usually aldosterone-producing adenoma [APA]) or bilateral (usually adrenal hyperplasia). Recently, somatic mutations in KCNJ5 (encoding a potassium channel) have been detected in about 40% of surgically removed APAs. The aim of this study was to screen for additional somatic mutations in KCNJ5 in a cohort of APAs removed from 87 Australian patients. Design and method: The full-length coding sequence and flanking regions of KCNJ5 in APA and adjacent cortex was resequenced. Functional changes caused by a novel mutation were studied by expressing wild-type (WT) or the mutant KCNJ5 channel in Xenopus oocytes (to examine electrophysiological effects) and transfecting empty GFP vector or the GFP-tagged mutant channel in human adrenocortical carcinoma (H295R) cells (to assess aldosterone release). Results: KCNJ5 mutations were detected in 37 APAs, and included the previously reported E145Q (n = 3), G151R (n = 20) and L168R (n = 13) mutations plus a novel 12-bp mutation, c.414–425dupCGCTTTCCTGTT (A139_F142dup) that duplicates the AFLP sequence just upstream of the selectivity filter. No mutations were found in adjacent cortices. On expression in Xenopus oocytes, the A139_F142dup mutation reduced the resting membrane potential and channel selectivity for potassium (K/Na permeability ratio 31 in WT KCNJ5 channels vs 7 in the A139_F142dup mutant). When transfected into H295R cells, A139_F142dup increased basal aldosterone release 2.3-fold over WT. This was not increased further by incubation with ATII. Clinically, the 54-year-old male from whom the mutation-bearing APA was removed had relatively severe PA with resistant hypertension, markedly elevated aldosterone/renin ratio (aldosterone 490 pmol/L, renin 2 mU/L, ratio 296) and an 11 mm left adrenal tumour on CT with lateralization to that side on adrenal venous sampling. Conclusions: Resequencing of a large Australian cohort of patients with APA further confirmed the major role of KCNJ5 somatic mutations in APA. The novel duplication mutation we report here has similar functional effects to the other mutations affecting the selectivity filter of the KCNJ5 channel with reduced membrane polarization, reduced selectivity to K and increased aldosterone release.