Biomedical subjects

Chou-Long Huang

Publications and source records attributed to Chou-Long Huang.

16 recordsLinked to original sources

PubMed2022

WNK1-OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy.

Lysine-deficient protein kinase-1 (WNK1) is critical for both embryonic angiogenesis and tumor-induced angiogenesis. However, the downstream effectors of WNK1 during these processes remain ambiguous. In this study, we identified that oxidative stress responsive 1b (osr1b) is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit ppp2r1bb. In addition, wnk1a and osr1b are upregulated in two liver cancer transgenic fish models: [tert x p53-/-] and [HBx,src,p53-/-,RPIA], while ppp2r1bb is downregulated in [tert x p53-/-]. Furthermore, using HUVEC endothelial cells co-cultured with HepG2 hepatoma cells, we confirmed that WNK1 plays a critical role in the induction of hepatoma cell migration in both endothelial cells and hepatoma cells. Moreover, overexpression of OSR1 can rescue the reduced cell migration caused by shWNK1 knockdown in HUVEC cells, indicating OSR1 is downstream of WNK1 in endothelial cells promoting hepatoma cell migration. Overexpression of PPP2R1A can rescue the increased cell migration caused by WNK1 overexpression in HepG2, indicating that PPP2R1A is a downstream effector in hepatoma. The combinatorial treatment with WNK1 inhibitor (WNK463) and OSR1 inhibitor (Rafoxanide) plus oligo-fucoidan via oral gavage to feed [HBx,src,p53-/-,RPIA] transgenic fish exhibits much more significant anticancer efficacy than Regorafenib for advanced HCC. Importantly, oligo-fucoidan can reduce the cell senescence marker-IL-1β expression. Furthermore, oligo-fucoidan reduces the increased cell senescence-associated β-galactosidase activity in tert transgenic fish treated with WNK1-OSR1 inhibitors. Our results reveal the WNK1-OSR1-PPP2R1A axis plays a critical role in both endothelial and hepatoma cells during tumor-induced angiogenesis promoting cancer cell migration. By in vitro and in vivo experiments, we further uncover the molecular mechanisms of WNK1 and its downstream effectors during tumor-induced angiogenesis. Targeting WNK1-OSR1-mediated anti-angiogenesis and anti-cancer activity, the undesired inflammation response caused by inhibiting WNK1-OSR1 can be attenuated by the combination therapy with oligo-fucoidan and may improve the efficacy.

Animals↗

PubMed2006

Role of with-no-lysine [K] kinases in the pathogenesis of Gordon's syndrome.

Gordon's syndrome, also known as pseudohypoaldosteronism type II (PHA II) or familial hypertension with hyperkalemia, is an autosomal-dominant disease characterized by hypertension, hyperkalemia, hyperchloremic metabolic acidosis, and normal glomerular filtration rate. Recent positional cloning has linked mutations of WNK1 and WNK4 to Gordon's syndrome. With-no-lysine [K] (WNK) kinases are a new family of large serine-threonine protein kinases with an atypical placement of the catalytic lysine. Here, we review the pathogenesis of PHA II based on current understanding of the actions of WNK1 and WNK4 on Na+ and K+ handling in the renal distal tubule.

Animals↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

Probing the effects of phosphoinositides on ion channels.

Ion channels are integral membrane proteins that control transmembrane ion fluxes to regulate membrane potential, cell excitability, and ion transport. Membrane phospholipids containing phosphoinositides have recently emerged as important regulators of many ion channels, including inward rectifier K+ channel, voltage-gated K+ and Ca2+ channels, transient receptor potential channels, and intracellular inositol-1,4,5-trisphosphate receptor ion channels. Discussed here are several methods for studying regulation of ion channels by phospholipids.

Animals↗

PubMed2005

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+.

Animals↗

PubMed2005

Conformational changes of pore helix coupled to gating of TRPV5 by protons.

The transient receptor potential channel TRPV5 constitutes the apical entry pathway for transepithelial Ca2+ transport. We showed that TRPV5 was inhibited by both physiological intra- and extracellular acid pH. Inhibition of TRPV5 by internal protons was enhanced by extracellular acidification. Similarly, inhibition by external protons was enhanced by intracellular acidification. Mutation of either an extra- or an intracellular pH sensor blunted the cross-inhibition by internal and external protons. Both internal and external protons regulated the selectivity filter gate. Using the substituted cysteine accessibility method, we found that intracellular acidification of TRPV5 caused a conformational change of the pore helix consistent with clockwise rotation along its long axis. Thus, rotation of pore helix caused by internal protons facilitates closing of TRPV5 by external protons. This regulation by protons likely contributes to pathogenesis of disturbances of Ca2+ transport in many diseased states. Rotation of pore helix may be a common mechanism for cross-regulation of ion channels by extra- and intracellular signals.

Amino Acid Sequence↗

PubMed2005

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.

Animals↗

PubMed2005

WNK1 activates SGK1 to regulate the epithelial sodium channel.

WNK (with no lysine [K]) kinases are serine-threonine protein kinases with an atypical placement of the catalytic lysine. Intronic deletions increase the expression of WNK1 in humans and cause pseudohypoaldosteronism type II, a form of hypertension. WNKs have been linked to ion carriers, but the underlying regulatory mechanisms are unknown. Here, we report a mechanism for the control of ion permeability by WNK1. We show that WNK1 activates the serum- and glucocorticoid-inducible protein kinase SGK1, leading to activation of the epithelial sodium channel. Increased channel activity induced by WNK1 depends on SGK1 and the E3 ubiquitin ligase Nedd4-2. This finding provides compelling evidence that this molecular mechanism contributes to the pathogenesis of hypertension in pseudohypoaldosteronism type II caused by WNK1 and, possibly, in other forms of hypertension.

Animals↗

PubMed2004

Inhibition of ROMK potassium channel by syntaxin 1A.

ROMK potassium channels are present in the cortical collecting duct (CCD) of the kidney and serve as apical exit pathways for K+ secretion in this nephron segment. K+ secretion in the CCD is regulated by multiple factors. In this study, we show that syntaxin 1A, but not syntaxin 3 or 4, inhibited whole cell ROMK currents in Xenopus laevis oocytes. Syntaxin 1A, but not syntaxin 3 or 4, interacted with the COOH-terminal cytoplasmic domain of ROMK in intro. Coexpression with synaptobrevin 2 reversed inhibition of whole cell ROMK currents by syntaxin 1A. In excised inside-out membranes of oocytes, application of fusion proteins containing the cytoplasmic region of syntaxin 1A to the cytoplasmic face caused a dose-dependent inhibition of ROMK. We further examined regulation of the K+ channels in the CCD by syntaxin 1A. Application of botulinum toxin C1 to the excised inside-out membranes of the CCD caused an increase in the activity of K+ channels. In contrast, application of toxin B had no effects. These results suggest that syntaxin 1A causes a tonic inhibition of the K+ channels in the apical membrane of the CCD. Binding of synaptobrevin 2 to syntaxin 1A during docking and fusion of transport vesicles to the plasma membranes of CCD may lead to activation of these channels.

Animals↗

PubMed2004

The N-terminal extracellular domain is required for polycystin-1-dependent channel activity.

Autosomal dominant polycystic kidney disease (PKD) is caused by mutation of polycystin-1 or polycystin-2. Polycystin-2 is a Ca(2+)-permeable cation channel. Polycystin-1 is an integral membrane protein of less defined function. The N-terminal extracellular region of polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now report that expression of polycystin-1 alone in Chinese hamster ovary (CHO) cells and in PKD2-null cells can confer Ca(2+)-permeable non-selective cation currents. Co-expression of a loss-of-function mutant of polycystin-2 in CHO cells does not reduce polycystin-1-dependent channel activity. A polycystin-1 mutant lacking approximately 2900 amino acids of the extracellular region is targeted to the cell surface but does not produce current. Extracellular application of antibodies against the immunoglobulin-like PKD domains reduces polycystin-1-dependent current. These results support the hypothesis that polycystin-1 is a surface membrane receptor that transduces the signal via changes in ionic currents.

Animals↗

PubMed2004

The transient receptor potential superfamily of ion channels.

ABSTRACT. The transient receptor potential (TRP) superfamily of proteins is cation-selective ion channels with six predicted transmembrane segments and intracellularly localized amino and carboxyl termini. Members of the TRP superfamily are identified on the basis of amino acid sequence and structural similarity and are classified into TRPC, TRPV, TRPM, TRPP, TRPN, and TRPML subfamilies. TRP channels are widespread and have diverse functions, ranging from thermal, tactile, taste, osmolar, and fluid flow sensing to transepithelial Ca2+ and Mg2+ transport. Mutations of TRP proteins produce many renal diseases, including Mg2+ wasting, hypocalcemia, and polycystic kidney diseases. This review focuses on recent advances in the understanding of their functions.

Animals↗

PubMed2003

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 approximately 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.

Amino Acid Substitution↗

PubMed2003

Dietary potassium restriction stimulates endocytosis of ROMK channel in rat cortical collecting duct.

ROMK potassium channels are present in the cortical collecting ducts (CCDs) of the kidney and serve as the exit pathways for K+ secretion in this nephron segment. Dietary K+ restriction reduces the abundance of ROMK in the kidney. We have previously shown that ROMK undergoes endocytosis via clathrin-coated vesicles in Xenopus laevis oocytes and in cultured cells. Here, we examined the effect of dietary K+ restriction on endocytosis of ROMK in CCDs using double-labeling immunofluorescent staining and confocal microscopic imaging in whole kidney sections as well as in individually isolated tubules. We found that ROMK abundance in kidney cortex and CCDs was reduced in rats fed a K+-restricted diet compared with rats fed the control K+ diet. In the control animals, ROMK staining was preferentially localized to the apical membrane of CCDs. Compared with control tubules, ROMK staining in CCDs was markedly shifted toward intracellular locations in animals fed a K+-deficient diet for 48 h. Some of the intracellular distribution of ROMK colocalized with an early endosomal marker, early endosomal antigen-1 or with a late endosomal/lysosomal marker, lysosomal membrane glycoprotein-120. These results suggest that K+ restriction reduces the abundance of ROMK in CCDs by increasing endocytosis and degradation of the channel protein. This decrease in the abundance of ROMK is likely important for maintaining K+ homeostasis during K+ deficiency.

Animals↗

PubMed2003

Protein kinase C inhibits ROMK1 channel activity via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism.

The activity of apical K(+) channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP(2)) and the cloned CCD K(+) channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP(2) and mutation of PKA sites reduces the affinity of ROMK1 for PIP(2). In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP(2) affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP(2) to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP(2) content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP(2) content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K(+) secretion in CCD by hormones that activate PKC.

Animals↗

PubMed2002

Structural determinants and specificities for ROMK1-phosphoinositide interaction.

We have recently reported that direct interaction between phosphatidylinositol bisphosphate (PIP(2)) and the COOH-terminal cytoplasmic domain of ROMK1 is important for opening of the channel. We identified arginine-188 of ROMK1 as a critical residue for this interaction. Here, we further report that substitution of a neutral amino acid for lysine-181, arginine-217, or lysine-218 decreases single-channel open probability for the full-conductance state and increases the frequency of opening at a subconductance state. Compared with wild-type ROMK1 channels, these substitution mutants also display an increased sensitivity to the block by anti-PIP(2) antibodies and to inhibition by intracellular protons. These results indicate that, like arginine-188, lysine-181, arginine-217, and lysine-218 are also involved in interactions with PIP(2) and are critical for ROMK1 to open at full conductance. Using synthetic phosphoinositides containing phosphates at different positions in the head group, we also examined the specificities of phosphoinositides in the regulation of ROMK1 channels. We found that phosphoinositides containing phosphate at both positions 4 and 5 of the inositol head group have the highest efficacy in activating ROMK1 channels. These results suggest that phosphatidylinositol 4,5-bisphosphate is likely the important phosphoinositide in the regulation of ROMK1 channels in a physiological membrane milieu.

Alanine↗

PubMed2002

Evidence for endocytosis of ROMK potassium channel via clathrin-coated vesicles.

ROMK channels are present in the cortical collecting ducts of kidney and are responsible for K(+) secretion in this nephron segment. Recent studies suggest that endocytosis of ROMK channels is important for regulation of K(+) secretion in cortical collecting ducts. We investigated the molecular mechanisms for endocytosis of ROMK channels expressed in Xenopus laevis oocytes and cultured Madin-Darby canine kidney cells. When plasma membrane insertion of newly synthesized channel proteins was blocked by incubation with brefeldin A, ROMK currents decreased with a half-time of ~6 h. Coexpression with the Lys44-->Ala dominant-negative mutant dynamin, but not wild-type dynamin, reduced the rate of reduction of ROMK in the presence of brefeldin A. Mutation of Asn371 to Ile in the putative NPXY internalization motif of ROMK1 abolished the effect of the Lys44-->Ala dynamin mutant on endocytosis of the channel. Coimmunoprecipitation study and confocal fluorescent imaging revealed that ROMK channels associated with clathrin coat proteins in Madin-Darby canine kidney cells. These results provide compelling evidence for endocytosis of ROMK channels via clathrin-coated vesicles.

Adenosine Triphosphate↗