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H Garty

Publications and source records attributed to H Garty.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of interactions between Nedd4 and beta and gammaENaC using surface plasmon resonance.

Cell surface expression of the epithelial Na(+) channel ENaC is regulated by the ubiquitin ligase Nedd4. Binding of the WW domains of Nedd4 to the PY region in the carboxy tails of beta and gammaENaC, results in channel ubiquitination and degradation. Kinetic analysis of these interactions has been done using surface plasmon resonance. Synthetic peptides corresponding to the PY regions of beta and gammaENaC were immobilized on a sensor chip and "real-time" kinetics of their binding to recombinant WW proteins was determined. Specificity of the interactions was established by competition experiment, as well as by monitoring effects of a point mutation known to impair Nedd4/ENaC binding. These data provides the first determination of association, dissociation and equilibrium constants for the interactions between WW2 and beta or gammaENaC.

Animals↗
PubMed2001

CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the gamma-subunit.

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the gamma-subunit of the Na,K-ATPase (NKA) that modulates the Na,K-pump function in the kidney. Here, we report that, similarly to gamma(a) and gamma(b) splice variants, the FXYD protein CHIF (corticosteroid-induced factor) is a type I membrane protein which is associated with NKA in renal tissue, and modulates the Na,K-pump transport when expressed in Xenopus oocytes. In contrast to gamma(a) and gamma(b), which both decrease the apparent Na+ affinity of the Na,K-pump, CHIF significantly increases the Na+ affinity and decreases the apparent K+ affinity due to an increased Na+ competition at external binding sites. The extracytoplasmic FXYD motif is required for stable gamma-subunit and CHIF interaction with NKA, while cytoplasmic, positively charged residues are necessary for the gamma-subunit's association efficiency and for CHIF's functional effects. These data document that CHIF is a new tissue-specific regulator of NKA which probably plays a crucial role in aldosterone-responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis.

Alternative Splicing↗
PubMed2001

Cloning and function of the rat colonic epithelial K+ channel KVLQT1.

KVLQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of KVLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to KVLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of KVLQT1 in crypt cells and surface epithelium. Expression of rKVLQT1 in Xenopus oocytes induced a typical delayed activated K+ current, that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that KVLQT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Membrane topology and immunolocalization of CHIF in kidney and intestine.

Corticosteroid hormone-induced factor (CHIF) is an aldosterone-induced gene, the function of which is yet unknown. It is specifically expressed in kidney collecting duct (CD) and distal colon and is upregulated by either Na+ deprivation or K+ loading. Hence, it may play a role in epithelial electrolyte transport. Previous studies have characterized regulation and tissue distribution of CHIF mRNA but provided no information on the protein itself. The present paper addresses this issue by using Western blotting, immunochemistry, and in vitro translation. CHIF is an approximately 8-kDa membranal protein, and protease digestion experiments suggest that its COOH tail faces the cell interior. The protein is abundant in distal colon, kidney medulla, and papilla but cannot be detected in a variety of other tissues. Confocal immunocytochemistry demonstrates that CHIF is present in the basolateral membrane of CD principal cells and distal colon surface cells, with occasional intracellular staining. Dexamethasone and low Na+ intake increase the abundance of CHIF. Unlike previous Northern data, induction of CHIF protein by low-Na+ intake was apparent not only in the distal colon but also in the kidney.

Animals↗
PubMed2001

In vitro phosphorylation of COOH termini of the epithelial Na(+) channel and its effects on channel activity in Xenopus oocytes.

Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na(+) channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the gamma-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on beta(S620) was phosphorylated as well. The possible role of gammaT630 and betaS620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na(+) and had a high basal Na(+) current. Thus mutating gammaT630 or betaS620 may limit the maximal channel activity achieved by a combination of sgk and low Na(+).

Animals↗
PubMed2000

Regulation of the epithelial Na+ channel by aldosterone: open questions and emerging answers.

Aldosterone is the principal adrenal steroid controlling Na+ retention in amphibians and mammalians. It acts primarily by increasing the apical Na+ permeability through activation of the epithelial Na+ channel (ENaC). The cellular events mediating the hormonal action are mostly unknown. Early studies have provided evidence that the hormone functions to activate or translocate pre-existing channels by a yet undefined mechanism. In addition, enhanced de novo channel synthesis appears to take place as well. The molecular cloning of the three ENaC subunits has provided new powerful tools for testing and confirming this hypothesis, as well as for characterizing mechanisms by which ENaC is regulated. Another important development is the recent identification of several cDNAs corresponding to aldosterone-induced and suppressed mRNAs. The study of these genes and their putative interactions with ENaC is likely to provide important clues to the mechanisms by which aldosterone controls the apical Na+ permeability of tight epithelia. This article reviews recent developments in the field that may lead to the elucidation of the mechanisms by which the hormone controls Na+ transport.

Aldosterone↗
PubMed2000

Regulation of sgk by aldosterone and its effects on the epithelial Na(+) channel.

Aldosterone is the major corticosteroid regulating Na(+) absorption in tight epithelia and acts primarily by activating the epithelial Na(+) channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na(+) current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514-2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgk mRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na(+) channel activity. A point mutation in the beta-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.

Aldosterone↗
PubMed1998

The ontogeny of the expression of K+ channel-like gene (CHIF) in the rat kidney papilla.

Recently, an IsK-like potassium (K+) channel corticosteroid-induced gene (CHIF) was cloned. A high-K+ diet enhances, while a low-K+ diet decreases the expression of this gene. The major expression of CHIF in the adult rat kidney is in the papilla, where it is constitutive, in contrast to its inducibility by corticosteroids and a low-salt diet in the rat colon. In order to further understand the ontogeny of K+ clearance, we studied the presence of CHIF in the kidney papilla in different stages of rat development. Total RNA from rat kidney papillae of 1- to 3-day pre-labor unborn offspring, 2- to 3-day-old newborns, 10-day-old, 6-week-old, and 43-week-old rats underwent northern hybridization for CHIF and the alpha-subunit of the Na+-K+-ATPase mRNA. Minor expression of CHIF mRNA was found in fetal and newborn rat papillae, while older rats showed an age-related increase in gene expression. The expression of the alpha-sub unit of the Na+-K+-ATPase was not age related. We conclude that CHIF is present in the rat kidney papilla and the expression is related to age. The relative deficiency of CHIF in the newborn may be one of the factors responsible for the reduced K+ clearance in is period.

Aging↗
PubMed1998

Differential regulation of ROMK expression in kidney cortex and medulla by aldosterone and potassium.

This study explores the role of K+ and aldosterone in the regulation of mRNA of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, in the rat kidney. K+ deficiency downregulated ROMK mRNA in cortex to 47.1 +/- 5.1% of control (P < 0.001) and in medulla to 56.1 +/- 3. 4% (P < 0.001). High-K+ diet slightly increased ROMK mRNA in medulla to 122 +/- 9% (P < 0.05 vs. control). Adrenalectomy (Adx) downregulated cortical ROMK mRNA to 30.7 +/- 6.8% (P < 0.001 vs. control), and increased it in medulla to 138 +/- 12.9% (P < 0.02 vs. control). In Adx rats, K+ deficiency decreased ROMK mRNA in cortex and medulla similar to intact rats. The alpha1- and beta1-Na-K-ATPase subunits were regulated in parallel to that of ROMK. In medulla, ROMK mRNA correlated with serum K+ concentration at R = 0.9406 (n = 6, P < 0.001) and alpha1-Na-K-ATPase mRNA at R = 0.9756 (n = 6, P < 0.001). ROMK2 also correlated with serum K+ concentration (R = 0.895; n = 6, P < 0.01). These results show that cortical ROMK expression is regulated by aldosterone and K+, whereas the medullary ROMK mRNA is regulated by serum K+.

Adrenalectomy↗
PubMed1997

An aldosterone regulated chicken intestine protein with high affinity to amiloride.

The pattern of chicken intestine amiloride-binding proteins was determined using the photoreactive amiloride analogue 2'-methoxy-5'-nitrobenzamil (NMBA) and a polyclonal anti-amiloride antibody. At 10(-7)M, NMBA inhibits approximately 62% of the Na+ channel activity. At this concentration the amiloride analogue labels a number of membrane proteins, and in particular a 40-45 kDa polypeptide denoted ABP40. Incorporation of NMBA into ABP40 could be prevented by a 100-fold excess of benzamil, but not by a 1000-fold excess of 5-(N-ethyl-N-isopropyl)-amiloride. Labeling of ABP40 was intense in membranes derived from salt-deprived chickens and approximately 5-fold weaker in membranes from salt-repleted animals. Because of its small size, ABP40 is not likely to be an avian Na+ channel subunit, yet this amiloride-binding protein could be involved in the response to aldosterone.

Aldosterone↗
PubMed1997

Cloning and induction by low NaCl intake of avian intestine Na+ channel subunits.

The alpha-subunit of the highly Na(+)-selective amiloride-blockable channel (ENaC) was cloned from chicken lower intestine. The deduced amino acid sequence of the avian clone exhibits -60% identity to the previously cloned mammalian and amphibian alpha-subunits. It also maintains the same hydropathy profile and structural motifs. These include two transmembrane domains separated by a large extracellular loop, four extracellular N-glycosylation sites, a cysteine-rich box in the extracellular domain, and a proline-rich stretch at the carboxy terminus. Xenopus oocytes injected with cRNA transcribed from this clone express a small amiloride-blockable Na+ conductance. Degenerate primers have been used to amplify two other related products. Sequence homology indicates that one of them is the beta-subunit, whereas the other appears to represent a closely related but different transcript. Regulation of the mRNA corresponding to these clones was examined in chickens fed normal and low-NaCl rations. The low-salt diet evoked an approximately fourfold increase in the abundance of mRNA coding for the alpha-subunit, presumably through an increase in plasma aldosterone. The beta- and "beta-like" transcripts were even more strongly affected. The current data provide additional information on sequence conservation in the growing ENaC family and demonstrate that the avian intestine channel is strongly induced by varying NaCl intake.

Amino Acid Sequence↗
PubMed1997

Differential regulation of CHIF mRNA by potassium intake and aldosterone.

The channel-inducing factor (CHIF) is an epithelial-specific transmembrane protein, which is induced by aldosterone in distal colon (but not in kidney) and can evoke K+ conductance in Xenopus oocytes. The current study examined the possibility that CHIF participates in maintaining K+ balance by assessing its regulation during variations in K+ intake. In adrenal-intact rats, high-K+ diet stimulated, whereas K+ deficiency downregulated, CHIF mRNA both in kidney and colon. The downregulation of CHIF observed in rats fed a low-K+ diet for different periods of time closely correlated with a decrease in plasma K+ but also with changes in aldosterone levels. To differentiate between the two, modulation of CHIF has been studied in adrenalectomized rats with and without corticosteroid supplementation. These experiments have demonstrated that a low-K+ intake suppresses CHIF mRNA, irrespective of aldosterone level. On the other hand, the upregulation evoked by a high-K+ load is apparent only in adrenal-intact rats. This is despite the fact that infusing rats with aldosterone and corticosterone does not increase the expression of this mRNA in kidney. These findings may suggest a role for CHIF in preserving K+ balance.

Acidosis↗
PubMed1997

Epithelial sodium channels: function, structure, and regulation.

The apical (outward-facing) membranes of high-resistance epithelia contain Na+ channels, traditionally identified by their sensitivity to block by the K(+)-sparing diuretic amiloride. Such channels have been characterized in amphibian skin and urinary bladder, renal collecting duct, distal colon, sweat and salivary glands, lung, and taste buds. They mediate the first step of active Na+ reabsorption and play a major role in the maintenance of electrolyte and water homeostasis in all vertebrates. In the past, these channels were classified according to their biophysical and pharmacological properties. The recent cloning of the three homologous channel subunits denoted alpha-, beta-, and gamma-epithelial Na+ channels (ENaC) has provided a molecular definition of at least one class of amiloride-blockable channels. Subsequent studies have established that ENaC is a major Na(+)-conducting pathway in both absorbing and secretory epithelia and is related to one type of channel involved in mechanosensation. This review summarizes the biophysical characteristics, molecular properties, and regulatory mechanisms of epithelial amiloride-blockable Na+ channels. Special emphasis is given to recent studies utilizing cloned ENaC subunits and purified amiloride-binding proteins.

Animals↗
PubMed1996

Aldosterone-induced increase in the abundance of Na+ channel subunits.

The highly selective, amilorideblockable Na+ channel is a major target to the natriferic action of the mineralocorticoid aldosterone. This rat epithelial Na+ channel (rENaC) has been recently cloned from colon and is composed of three homologous subunits denoted alpha-, beta-, and gamma-rENaC (C. M. Canessa, L. Schild, G. Buell, B. Thorens, L. Gautschi, J.-D. Horisberger, and B. C. Rossier. Nature Lond. 367: 463-467, 1994). We have tested the effects of corticosteroids on the abundance of mRNA coding for each subunit in kidney cortex and distal colon. Chronic treatment of rats with aldosterone or dexamethasone evoked in kidney cortex a small induction of alpha-rENaC and no change in beta- and gamma-rENaC. In distal colon, however, beta- and gamma-rENaC were strongly induced by either aldosterone or dexamethasone, whereas alpha-rENaC was constitutively expressed. Most of the aldosterone-induced increase in beta- and gamma-rENaC mRNA took place during 3-24 h after plasma aldosterone was elevated. A similar differential induction of rENaC subunits in kidney and colon was also evoked by a Na(+)-free diet. The effects of salt deprivation were reversed by resalinating rats with a half time of < 2 h, suggesting a high turnover rate of at least beta- and gamma-rENaC. The data are consistent with the possibility that induction of channel subunits contributes to the chronic but not the acute response to aldosterone in the colon. Such a mechanism is not likely to play a major role in cortical collecting ducts.

Aldosterone↗
PubMed1996

Cellular localization and regulation of CHIF in kidney and colon.

Channel inducing factor (CHIF) is a novel cDNA recently cloned from a rat distal colon cDNA library of dexamethasone-treated animals. While its expression in Xenopus oocytes evokes a potassium channel activity similar to that induced by Isk (minK), its cellular role is not clear. CHIF exhibits significant homologies with proteins that are putatively regulatory (phospholemman, gamma-subunit of Na(+)-K(+)-ATPase, Mat-8) while it differs from the small-conductance potassium channel Isk. We have studied the tissue specificity of CHIF expression in rat by in situ hybridization. CHIF is selectively present in the distal parts of the nephron (medullary and papillary collecting ducts and end portions of cortical collecting tubule) and in the epithelial cells of the distal colon. No expression of CHIF was found in renal proximal tubule, loop of Henle and distal tubule, proximal colon, small intestine, lung, choroid plexus, salivary glands, or brain. To gain some insight into CHIF function, we have investigated, using in situ hybridization and ribonuclease protection assay, whether CHIF mRNA expression could be altered in some situations. In the distal colon, corticosteroid hormones, sodium restriction, low-potassium diet, and metabolic acidosis significantly increased CHIF mRNA expression. In the kidney, metabolic acidosis was the only condition that showed an increase in CHIF mRNA expression. Some of these treatments also altered the expression of the colonic H(+)-K(+)-ATPase mRNA. In summary, CHIF mRNA is selectively expressed in the medullary collecting duct of the kidney and in the epithelium of the distal colon; its expression varies differently in these two target tissues after alterations in corticosteroid status, potassium depletion, and metabolic acidosis. The precise cell-specific functions of CHIF remain to be established.

Animals↗
PubMed1996

Inhibition of amiloride-sensitive Na+ channel by isothiouronium derivatives.

The effects on the amiloride-blockable Na+ channel of a family of recently synthesized isothiouronium derivatives were measured in plasma membrane vesicles from rat distal colon. Some of these derivatives act as high-affinity Na(+)-like antagonists on the Na(+)-K(+)-adenosinetriphosphatase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium methyl)-benzene tribromide (Br-TITU), was found to be a potent blocker of the Na+ channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated 22Na+ uptake, with an inhibition constant of 94 +/- 39 nM. The inhibition observed is specific and reversible. 1,3-Dibromo-2,4,6-tris(isothiouronium methyl)benzene tribromide and Br-TITU derivatives with methyl and phenyl substitutions on the isothiouronium moiety were much less effective blockers. Incubation of cells with Br-TITU at alkaline (but not neutral) pH produces irreversible inactivation of channels, possibly due ot covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TITU may be a useful reagent in identifying essential residues of the channel protein.

Amiloride↗
PubMed1996

Aldosterone induction and epithelial distribution of CHIF.

CHIF is a recently cloned, corticosteroid-induced gene which evokes K+ channel activity in oocytes (B. Attali, H. Latter, N. Rachamim, and H. Garty. Proc. Natl. Acad. Sci. USA 92: 6092-6096, 1995). To further characterize the possible role of this gene in epithelial ion transport, we have studied its epithelial distribution and hormonal induction. Northern hybridizations indicate that the zonal distribution of CHIF mRNA in kidney is: papilla >>medulla>> cortex. High levels of CHIF were also detected in a primary culture from inner medullary collecting duct (IMCD). Perfusing rats with < 20 nM aldosterone through osmotic minipumps evoked a 22.4 +/- 1.9-fold increase in colonic CHIF. A significant increase was observed 3 h after administrating the corticosteroid, but maximal response was detected only after a 72-h incubation. This response appears to be mineralocorticoid specific; perfusing or injecting rats with maximal doses of dexamethasone did not evoke a further increase in CHIF mRNA. In contrast, high levels of CHIF are expressed in kidney papilla and IMCD primary culture, irrespective of corticosteroid treatment. Thus, like the apical Na+ channel and the H(+)-K(+)-adenosinetriphosphatase, CHIF is mineralocorticoid induced in the colon but constitutively expressed in kidney.

Aldosterone↗