Chou Long Huang

Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gβγ

Inward rectifier K+ channels, which modulate electrical activity in many cell types, are regulated by protein kinases,, guanine-nucleotide-binding proteins (G proteins) and probably actin cytoskeleton. Generation of phosphatidylinositol 4,5-bisphosphate (PIP2) by ATP-dependent lipid kinases is known to activate inward rectifier K+ channels in cardiac membrane patches. Herewe report that several cloned inward rectifier K+ channels directly bind PIP2, and that this binding correlates with channel activity. Application of ATP or PIP2 liposomes activates the cloned channels. Stabilized by lipid phosphatase inhibitors, PIP2 antibodies potently inhibit each channel with a unique rate (GIRK1/4 (refs 3-5) ≈ GIRK2 (ref. 6) ≫ IRK1 (ref. 10) ≈ ROMK (ref. 11)). Consistent with the faster dissociation of PIP2 from the GIRK channels, the carboxy terminus of GIRK1 binds 3H-PIP2 liposomes more weakly than does that of IRK1 or ROMK1. Mutation of a conserved arginine to glutamine at position 188 reduces the ability of ROMK1 to bind PIP2 and increases its sensitivity to inhibition by PIP2 antibodies. Interactions between GIRK channels and PIP2 are modulated by the βγ subunits of the G protein (Gβγ). When GIRK1/4 channels are allowed to run down completely, they are not activated by addition of Gβγ alone, but application of PIP2 activates them in minutes without Gβγ and in just seconds with Gβγ. Finally, coexpression of Gβγ with GIRK channels slows the inhibition of K+ currents by PIP2 antibodies by more than 10-fold. Thus Gβγ activates GIRK channels by stabilizing interactions between PIP2 and the K+ channel.

Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1

Klotho is a mammalian senescence-suppression protein that has homology with glycosidases. The extracellular domain of Klotho is secreted into urine and blood and may function as a humoral factor. Klotho-deficient mice have accelerated aging and imbalance of ion homeostasis. Klotho treatment increases cell-surface abundance of the renal epithelial Ca2+ channel TRPV5 by modifying its N-linked glycans. However, the precise sugar substrate and mechanism for regulation by Klotho is not known. Here, we report that the extracellular domain of Klotho activates plasma-membrane resident TRPV5 through removing terminal sialic acids from their glycan chains. Removal of sialic acids exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 lattice at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. Knockdown of β-galactoside α2,6-sialyltransferase (ST6Gal-1) by RNA interference, but not other sialyltransferases, in a human cell line prevents the regulation by Klotho. Moreover, the regulation by Klotho is absent in a hamster cell line that lacks endogenous ST6Gal-1, but is restored by forced expression of recombinant ST6Gal-1. Thus, Klotho participates in specific removal of α2,6-linked sialic acids and regulates cell surface retention of TRPV5 through this activity. This action of Klotho represents a novel mechanism for regulation of the activity of cell-surface glycoproteins and likely contributes to maintenance of calcium balance by Klotho.

Antagonistic regulation of ROMK by long and kidney-specific WNK1 isoforms

WNK kinases are serine-threonine kinases with an atypical placement of the catalytic lysine. Intronic deletions with increased expression of a ubiquitous long WNK1 transcript cause pseudohypoaldosteronism type 2 (PHA II), characterized by hypertension and hyperkalemia. Here, we report that long WNK1 inhibited ROMK1 by stimulating its endocytosis. Inhibition of ROMK by long WNK1 was synergistic with, but not dependent on, WNK4. A smaller transcript of WNK1 lacking the N-terminal 1-437 amino acids is expressed highly in the kidney. Whether expression of the KS-WNK1 (kidney-specific, KS) is altered in PHA II is not known. We found that KS-WNK1 did not inhibit ROMK1 but reversed the inhibition of ROMK1 caused by long WNK1. Consistent with the lack of inhibition by KS-WNK1, we found that amino acids 1-491 of the long WNK1 were sufficient for inhibiting ROMK. Dietary K+ restriction decreases ROMK abundance in the renal cortical-collecting ducts by stimulating endocytosis, an adaptative response important for conservation of K+ during K+ deficiency. We found that K+ restriction in rats increased whole-kidney transcript of long WNK1 while decreasing that of KS-WNK1. Thus, KS-WNK1 is a physiological antagonist of long WNK1. Hyperkalemia in PHA II patients with PHA II mutations may be caused, at least partially, by increased expression of long WNK1 with or without decreased expression of KS-WNK1.

Mechanism of Hypokalemia in Magnesium Deficiency

Magnesium deficiency is frequently associated with hypokalemia. Concomitant magnesium deficiency aggravates hypokalemia and renders it refractory to treatment by potassium. Herein is reviewed literature suggesting that magnesium deficiency exacerbates potassium wasting by increasing distal potassium secretion. A decrease in intracellular magnesium, caused by magnesium deficiency, releases the magnesium-mediated inhibition of ROMK channels and increases potassium secretion. Magnesium deficiency alone, however, does not necessarily cause hypokalemia. An increase in distal sodium delivery or elevated aldosterone levels may be required for exacerbating potassium wasting in magnesium deficiency.

Regulation of Renal Outer Medullary Potassium Channel and Renal K+ Excretion by Klotho

Klotho is an aging-suppression protein predominantly expressed in kidney, parathyroid glands, and choroids plexus of the brain. The extracellular domain of Klotho, a type-1 membrane protein, is secreted into urine and blood and may function as an endocrine or paracrine hormone. The functional role of Klotho in the kidney remains largely unknown. Recent studies reported that treatment by the extracellular domain of Klotho (KLe) increases cell-surface abundance of transient receptor potential vanilloid type isoform 5, an epithelial Ca2+ channel critical for Ca2+ reabsorption in the kidney. Whether Klotho regulates surface expression of other channels in the kidney is not known. Here, we report that KLe treatment increases the cell-membrane abundance of the renal K+ channel renal outer medullary potassium channel 1 (ROMK1) by removing terminal sialic acids from N-glycan of the channel. Removal of sialic acids exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 at the extracellular surface prevents clathrin-mediated endocytosis of ROMK1 and leads to accumulation of functional channel on the plasma membrane. Intravenous administration of KLe increases the level of Klotho in urine and increases urinary excretion of K+. These results suggest that Klotho may have a broader function in the regulation of ion transport in the kidney.

Cardioprotection by Klotho through downregulation of TRPC6 channels in the mouse heart

Klotho is a membrane protein predominantly produced in the kidney that exerts some anti-ageing effects. Ageing is associated with an increased risk of heart failure; whether Klotho is cardioprotective is unknown. Here we show that Klotho-deficient mice have no baseline cardiac abnormalities but develop exaggerated pathological cardiac hypertrophy and remodeling in response to stress. Cardioprotection by Klotho in normal mice is mediated by downregulation of TRPC6 channels in the heart. We demonstrate that deletion of Trpc6 prevents stress-induced exaggerated cardiac remodeling in Klotho-deficient mice. Furthermore, mice with heart-specific overexpression of TRPC6 develop spontaneous cardiac hypertrophy and remodeling. Klotho overexpression ameliorates cardiac pathologies in these mice and improves their long-term survival. Soluble Klotho present in the systemic circulation inhibits TRPC6 currents in cardiomyocytes by blocking phosphoinositide-3-kinase-dependent exocytosis of TRPC6 channels. These results provide a new perspective on the pathogenesis of cardiomyopathies and open new avenues for treatment of the disease.

PIP2 activates TRPV5 and releases its inhibition by intracellular Mg2

The transient receptor potential type V5 channel (TRPV5) is a Ca2+-selective TRP channel important for epithelial Ca2+ transport. Intracellular Mg2+ causes a fast voltage-dependent block of the TRPV5 channel by binding to the selectivity filter. Here, we report that intracellular Mg2+ binding to the selectivity filter of TRPV5 also causes a slower reversible conformational change leading to channel closure. We further report that PIP2 activates TRPV5. Activation of TRPV5 by PIP2 is independent of Mg2+. Yet, PIP2 decreases sensitivity of the channel to the Mg2+-induced slow inhibition. Mutation of aspartate-542, a critical Mg2+-binding site in the selectivity filter, abolishes Mg2+-induced slow inhibition. PIP2 has no effects on Mg2+-induced voltage-dependent block. Thus, PIP2 prevents the Mg2+-induced conformational change without affecting Mg2+ binding to the selectivity filter. Hydrolysis of PIP2 via receptor activation of phospholipase C sensitizes TRPV5 to the Mg2+-induced slow inhibition. These results provide a novel mechanism for regulation of TRP channels by phospholipase C–activating hormones via alteration of the sensitivity to intracellular Mg2+.

Mechanism and molecular determinant for regulation of rabbit transient receptor potential type 5 (TRPV5) channel by extracellular pH.

The transient receptor potential type 5 (TRPV5) channel is present in kidney and intestine and important for transepithelial (re)absorption of calcium in these tissues. We report that in whole-cell patch clamp recording extracellular acidification inhibited rabbit TRPV5 with apparent pKa ∼6.55. The two extracellular loops between the fifth and sixth transmembrane segments of TRPV5 presumably form part of the outer opening of the pore and likely are important in binding and regulation by external protons. We found that mutation of glutamate 522 to glutamine (E522Q) decreased the sensitivity of the channel to extracellular acidification. Mutations of other titratable amino acids within the two extracellular loops to non-titratable amino acids had no effect on pH sensitivity. Substitutions of aspartate or other titratable amino acids for glutamate 522 conferred an increase in pH sensitivity. The pH sensitivity mediated by glutamate 522 was independent of extracellular or intracellular Mg2+. Single channel analysis revealed that extracellular acidification reduced single channel conductance as well as open probability of the wild type channel. In contrast to wild type channel, extracellular acidification did not reduce open probability for E522Q mutant. Methanethiosulfonate reagents inhibited the activity of glutamine 522 to cysteine mutant channel with a reaction rate constant approaching that with free thiols in solution, suggesting that glutamate 522 is located on the surface of the channel. These data suggest that glutamate 522 of the rabbit TRPV5 is a “pH sensor,” and extracellular protons inhibit TRPV5 likely by altering conformation of the channel protein.

WNK1 activates SGK1 by a phosphatidylinositol 3-kinase-dependent and non-catalytic mechanism

WNK1 (with no lysine (K) 1) is a protein-serine/threonine kinase with a unique catalytic site organization. Deletions in the first intron of the WNK1 gene were found in a group of hypertensive patients with pseudohypoaldosteronism type II. No changes in coding sequence of WNK1 were found, but its expression was increased severalfold. We have been investigating actions of WNK1 and have found that WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1, which impacts membrane expression of the epithelial sodium channel. Here we explore the role of WNK1 in SGK1 regulation. Activation of SGK1 by WNK1 is blocked by phosphatidylinositol 3-kinase inhibitors. Neither the catalytic activity nor the kinase domain of WNK1 is required; rather the N-terminal 220 residues of WNK1 are necessary and sufficient to activate SGK1. Phosphorylation of WNK1 on Thr-58 contributes to SGK1 activation. Finally, we show that WNK1 is required for the activation of SGK1 by insulin-like growth factor 1.

Soluble Klotho Protects against Uremic Cardiomyopathy Independently of Fibroblast Growth Factor 23 and Phosphate

Cardiac hypertrophy occurs in up to 95% of patients with CKD and increases their risk for cardiovascular death. In the kidney, full-length membranous Klotho forms the coreceptor for fibroblast growth factor 23 (FGF23) to regulate phosphate metabolism. The prevailing view is that the decreased level of Klotho in CKD causes cardiomyopathy through increases in serum FGF23 and/or phosphate levels. However, we reported recently that soluble Klotho protects against cardiac hypertrophy by inhibiting abnormal calcium signaling in the heart. Here, we tested whether this protective effect requires changes in FGF23 and/or phosphate levels. Heterozygous Klotho-deficient CKD mice exhibited aggravated cardiac hypertrophy compared with wild-type CKD mice. Cardiac magnetic resonance imaging studies revealed that Klotho-deficient CKD hearts had worse functional impairment than wild-type CKD hearts. Normalization of serum phosphate and FGF23 levels by dietary phosphate restriction did not abrogate the aggravated cardiac hypertrophy observed in Klotho-deficient CKD mice. Circulating levels of the cleaved soluble ectodomain of Klotho were lower in wild-type CKD mice than in control mice and even lower in Klotho-deficient CKD mice. Intravenous delivery of a transgene encoding soluble Klotho ameliorated cardiac hypertrophy in Klotho-deficient CKD mice. These results suggest that the decreased level of circulating soluble Klotho in CKD is an important cause of uremic cardiomyopathy independent of FGF23 and phosphate, opening new avenues for treatment of this disease.