WNK4 regulates the balance between renal NaCl reabsorption and K+ secretion.

A key question in systems biology is how diverse physiologic processes are integrated to produce global homeostasis. Genetic analysis can contribute by identifying genes that perturb this integration. One system orchestrates renal NaCl and K+ flux to achieve homeostasis of blood pressure and serum K+ concentration. Positional cloning implicated the serine-threonine kinase WNK4 in this process; clustered mutations in PRKWNK4, encoding WNK4, cause hypertension and hyperkalemia (pseudohypoaldosteronism type II, PHAII) by altering renal NaCl and K+ handling. Wild-type WNK4 inhibits the renal Na-Cl cotransporter (NCCT); mutations that cause PHAII relieve this inhibition. This explains the hypertension of PHAII but does not account for the hyperkalemia. By expression in Xenopus laevis oocytes, we show that WNK4 also inhibits the renal K+ channel ROMK. This inhibition is independent of WNK4 kinase activity and is mediated by clathrin-dependent endocytosis of ROMK, mechanisms distinct from those that characterize WNK4 inhibition of NCCT. Most notably, the same mutations in PRKWNK4 that relieve NCCT inhibition markedly increase inhibition of ROMK. These findings establish WNK4 as a multifunctional regulator of diverse ion transporters; moreover, they explain the pathophysiology of PHAII. They also identify WNK4 as a molecular switch that can vary the balance between NaCl reabsorption and K+ secretion to maintain integrated homeostasis.

Two-pore domain K+ channels-molecular sensors.

Two-pore domain K(+) (K2P) channels have been cloned from a variety of species and tissues. They have been characterised biophysically as a 'background' K(+)-selective conductance and are gated by pH, stretch, heat, coupling to G-proteins and anaesthetics. Whilst their precise physiological function is unknown, they are likely to represent an increasingly important family of membrane proteins.

Selectivity and interactions of Ba2+ and Cs+ with wild-type and mutant TASK1 K+ channels expressed in Xenopus oocytes.

The acid-sensitive K(+) channel, TASK1 is a member of the K(+)-selective tandem-pore domain (K2P) channel family. Like many of the K2P channels, TASK1 is relatively insensitive to conventional channel blockers such as Ba(2+). In this paper we report the impact of mutating the pore-neighbouring histidine residues, which are involved in pH sensing, on the sensitivity to blockade by Ba(2+) and Cs(+); additionally we compare the selectivity of these channels to extracellular K(+), Na(+) and Rb(+). H98D and H98N mutants showed reduced selectivity for K(+) over both Na(+) and Rb(+), and significant permeation of Rb(+). This enhanced permeability must reflect changes in the structure or flexibility of the selectivity filter. Blockade by Ba(2+) and Cs(+) was voltage-dependent, indicating that both ions block within the pore. In 100 mm K(+), the K(D) at 0 mV for Ba(2+) was 36 +/- 10 mm (n = 6), whilst for Cs(+) it was 20 +/- 6.0 mm (n = 5). H98D was more sensitive to Ba(2+) than the wild-type (WT); in addition, the site at which Ba(2+) appears to bind was altered (WT: delta, 0.64 +/- 0.16, n = 6; H98D: delta, 0.16 +/- 0.03, n = 5, statistically different from WT; H98N: delta, 0.58 +/- 0.09, not statistically different from WT). Thus, the pore-neighbouring residue H98 contributes not only to the pH sensitivity of TASK1, but also to the structure of the conduction pathway.

Phosphorylation-regulated endoplasmic reticulum retention signal in the renal outer-medullary K+ channel (ROMK).

The renal outer-medullary K+ channel (ROMK; Kir1.1) mediates K+ secretion in the renal mammalian nephron that is critical to both sodium and potassium homeostasis. The posttranscriptional expression of ROMK in the plasma membrane of cells is regulated by delivery of protein from endoplasmic reticulum (ER) to the cell surface and by retrieval by dynamin-dependent endocytic mechanisms in clathrin-coated pits. The S44 in the NH(2) terminus of ROMK1 can be phosphorylated by PKA and serum- and glucocorticoid-inducible kinase-1, and this process increases surface expression of functional channels. We present evidence that phosphorylation of S44 modulates channel expression by increasing its cell surface delivery consequent to suppression of a COOH-terminal ER retention signal. This phosphorylation switch of the ER retention signal could provide a pool of mature and properly folded channels for rapid delivery to the plasma membrane. The x-ray crystal structures of inward rectifier K+ channels have shown a close apposition of the NH(2) terminus with the distal COOH terminus of the adjacent subunit in the channel homotetramer, which is important to channel gating. Thus, NH(2)-terminal phosphorylation modifying a COOH-terminal ER retention signal in ROMK1 could serve as a checkpoint for proper subunit folding critical to channel gating.

Determinants of pH sensing in the two-pore domain K(+) channels TASK-1 and -2.

TASK-1 and -2 are members of the two-pore domain potassium (K(+)) channel family and are sensitive to changes in extracellular pH. The effects of mutating charged, extracellular-facing residues in TASK-1 and -2 were studied in Xenopusoocytes by two-electrode voltage clamp. Hydrogen ion block was independent of voltage with K(d) values of 149+/-17.9 nM [H(+)] ( n=6) and 5.76+/-1.23 nM [H(+)] ( n=7) for TASK-1 and -2, respectively. Compared to wild-type TASK-1, H72N, H98N, H98D and K210N displayed significant shifts in their K(d) values for hydrogen ion block ([H(+)]; 110+/-9.80 nM, 737+/-170 nM, 321+/-85.9 nM and 267+/-9.92 nM, respectively, n=6 each, P<0.05). Although significantly reducing its pH sensitivity, mutation of H98 in TASK-1 did not abolish pH sensitivity; this implies that H98 is not the only residue or domain involved in pH sensing of TASK-1. TASK-2 does not possess a histidine residue at the homologous position. However, the inclusion of such a residue failed to produce the expected increase in pH sensitivity; instead, a slight decrease was observed. Despite their structural homology and common sensitivity to pH, the TASK family of K(+) channels apparently has diverse pH-sensing mechanisms.