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1.
Tushare Jinadasa El?d Z. Szabó Masayuki Numata John Orlowski 《The Journal of biological chemistry》2014,289(30):20879-20897
Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H+-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na+/H+ exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress. 相似文献
2.
Increased recycling and elevated cell surface expression of receptors serve as a mechanism for persistent receptor-mediated signaling. We show that the neuron-enriched Na+/H+ exchanger NHE5 is abundantly expressed in C6 glioma cells and plays an important part in regulating cell surface expression of the receptor tyrosine kinases MET and EGF receptor. NHE5 is associated with transferrin receptor (TfR)- and Rab11-positive recycling endosomal membranes, and NHE5 knockdown by short hairpin RNA significantly elevates pH of TfR-positive recycling endosomes. We present evidence that NHE5 facilitates MET recycling to the plasma membrane, protects MET from degradation, and modulates HGF-induced phosphatidylinositol-3-kinase and mitogen-activated protein kinase signaling. Moreover, NHE5 depletion abrogates Rac1 and Cdc42 signaling and actin cytoskeletal remodeling. We further show that NHE5 knockdown impairs directed cell migration and causes loss of cell polarity. Our study highlights a possible role of recycling endosomal pH in regulating receptor-mediated signaling through vesicular trafficking. 相似文献
3.
Wormmeester Louktje; De Medina Fermin Sanchez; Kokke Freddy; Tse Chung-Ming; Khurana Seema; Bowser Joellyn; Cohen Michael E.; Donowitz Mark 《American journal of physiology. Cell physiology》1998,274(5):C1261
Intestinal neutral NaCl absorption, which is made up ofbrush-border (BB)Na+/H+exchange linked to BBCl/HCO3exchange, is up- and downregulated as part of digestion and diarrhealdiseases. Glucocorticoids stimulate ileal NaCl absorption and BBNa+/H+exchange. Intestinal BB contains twoNa+/H+exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbitileal BBNa+/H+exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BBNa+/H+exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ~50% to ilealNa+/H+exchange. Glucocorticoids (methylprednisolone) increase BBNa+/H+exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times),without an effect on NHE2 activity. Thus ileal BBNa+/H+exchange in animals treated with glucocorticoids is 69% via NHE3. Aquantitative Western analysis for NHE3 was developed, using as aninternal standard a fusion protein of the COOH-terminal 85 amino acidsof NHE3 and maltose binding protein. Glucocorticoid treatment increasedthe amount of BB NHE3. The quantitative Western analysis showed thatNHE3 makes up 0.018% of ileal BB protein in control rabbits and0.042% (2.3 times as much) in methylprednisolone-treated rabbits.Methylprednisolone treatment did not alter the amount of ileal BB NHE2protein. NHE3 turnover number was estimated to be 458 cycles/s underbasal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thusmethylprednisolone stimulates ileal BBNa+/H+exchange activity only by an effect on NHE3 and not on NHE2; it does soprimarily by increasing the amount of BB NHE3, although it alsoincreases the NHE3 turnover number. 相似文献
4.
The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na+/H+ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of 22Na+ study in the presence of 50 microM HOE-694 revealed that Na+ uptake mediated by NHE3 was increased approximately 88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na+ uptake by stimulating NHE3 mRNA and protein content. 相似文献
5.
Alexander RT Malevanets A Durkan AM Kocinsky HS Aronson PS Orlowski J Grinstein S 《The Journal of biological chemistry》2007,282(10):7376-7384
The epithelial Na(+)/H(+) exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them. 相似文献
6.
Vincent Chaptal Michela Ottolia Gabriel Mercado-Besserer Debora A. Nicoll Kenneth D. Philipson Jeff Abramson 《The Journal of biological chemistry》2009,284(22):14688-14692
The mammalian Na+/Ca2+ exchanger, NCX1.1, serves as
the main mechanism for Ca2+ efflux across the sarcolemma following
cardiac contraction. In addition to transporting Ca2+, NCX1.1
activity is also strongly regulated by Ca2+ binding to two
intracellular regulatory domains, CBD1 and CBD2. The structures of both of
these domains have been solved by NMR spectroscopy and x-ray crystallography,
greatly enhancing our understanding of Ca2+ regulation.
Nevertheless, the mechanisms by which Ca2+ regulates the exchanger
remain incompletely understood. The initial NMR study showed that the first
regulatory domain, CBD1, unfolds in the absence of regulatory Ca2+.
It was further demonstrated that a mutation of an acidic residue involved in
Ca2+ binding, E454K, prevents this structural unfolding. A
contradictory result was recently obtained in a second NMR study in which
Ca2+ removal merely triggered local rearrangements of CBD1. To
address this issue, we solved the crystal structure of the E454K-CBD1 mutant
and performed electrophysiological analyses of the full-length exchanger with
mutations at position 454. We show that the lysine substitution replaces the
Ca2+ ion at position 1 of the CBD1 Ca2+ binding site and
participates in a charge compensation mechanism. Electrophysiological analyses
show that mutations of residue Glu-454 have no impact on Ca2+
regulation of NCX1.1. Together, structural and mutational analyses indicate
that only two of the four Ca2+ ions that bind to CBD1 are important
for regulating exchanger activity.Cardiac contraction/relaxation relies upon Ca2+ fluxes across
the plasma membrane (sarcolemma) of cardiomyocytes. Rapid Ca2+
influx (primarily through L-type Ca2+ channels) triggers the
release of additional Ca2+ from the sarcoplasmic reticulum
(SR),4 resulting in
cardiomyocyte contraction. Removal of cytosolic Ca2+ by reuptake
into the SR (through the SR Ca2+-ATPase) and expulsion from the
cell (primarily through the Na+/Ca2+ exchanger, NCX1.1)
results in relaxation (1).
Altered Ca2+ cycling is observed in a number of pathophysiological
situations including ischemia, hypertrophy, and heart failure
(2). Understanding the function
and regulation of NCX1.1 is thus of fundamental importance to understand
cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na+
gradient to extrude Ca2+ in a ratio of three Na+ ions to
one Ca2+ ion (3). In
addition to transporting both Na+ and Ca2+, NCX1.1 is
also strongly regulated by these two ions. Intracellular Na+ can
induce NCX1.1 to enter an inactivated state, whereas Ca2+ bound to
regulatory sites removes Na+-dependent inactivation and also
activates Na+/Ca2+ exchange
(3). These regulatory sites are
located on a large cytoplasmic loop (∼500 residues located between
transmembrane helices V and VI) containing two calcium binding domains (CBD1
and CBD2), which sense cytosolic Ca2+ levels. We have previously
shown that Ca2+ binding to the primary site in CBD2 is required for
full exchange regulation (4);
CBD1, however, is a site of higher affinity and appears to dominate the
activation of exchange activity by Ca2+.Both CBDs have an immunoglobulin fold formed from two antiparallel β
sheets generating a β sandwich with a differing number of Ca2+
ions coordinated at the tip of the domain
(4,
5). CBD1 binds four
Ca2+ ions, whereas CBD2 binds only two Ca2+ ions. An
initial NMR study revealed a local unfolding of the upper portion of CBD1 upon
release of Ca2+ (6).
In contrast, CBD2 did not display an unfolding response upon Ca2+
removal. A comparative analysis between CBDs revealed a difference in charge
at residues in equivalent positions near the Ca2+ coordination
site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of
CBD1 upon Ca2+ removal was alleviated by reversing the charge of
the acidic residue (E454K) involved in Ca2+ coordination
(6). Previously, we solved the
structures of the Ca2+-bound and -free conformations of CBD2 and
revealed a charge compensation mechanism involving Lys-585
(4). The positively charged
lysine residue assumes the position of one of the Ca2+ ions upon
Ca2+ depletion, permitting CBD2 to retain its overall fold
(4). A similar phenomenon is
predicted to take place in E454K-CBD1 mutant. In addition, Hilge et
al. (6) showed that the
E454K mutation of CBD1 decreases Ca2+ affinity to a level similar
to that of CBD2 and suggested that the E454K mutation would cause the loss of
primary regulation of NCX1.1 by CBD1.The significance of some of these observations is unclear as a recent NMR
study (7) of CBD1 under more
physiologically relevant conditions revealed no significant alteration in
tertiary structure in the absence of Ca2+. It was hypothesized that
Ca2+ binding induces localized conformational and dynamic changes
involving several of the binding site residues. To clarify this issue, we
solved the crystal structure of the E454K-CBD1 mutant and examined the
functional effects of different CBD1 mutations in the full-length NCX1.1. The
results indicate that charge compensation is indeed provided by the residue
Lys-454 to replace one Ca2+, whereas the overall E454K-CBD1
structure is only slightly perturbed. The charge compensation, however, has no
impact on Ca2+ regulation of NCX1.1. 相似文献
7.
Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney 总被引:2,自引:0,他引:2
Bookstein Crescence; Xie Yue; Rabenau Karen; Musch Mark W.; McSwine Rebbecca L.; Rao Mrinalini C.; Chang Eugene B. 《American journal of physiology. Cell physiology》1997,273(5):C1496
We present evidence that tissue distribution of two highlyconservedNa+/H+exchanger isoforms, NHE2 and NHE4, differs significantly from previously published reports. Riboprobes unique to each of these antiporters, from 5' (noncoding and coding) and 3' codingregions, were used to analyze mRNA from adult rat kidney and intestine by ribonuclease protection assay and in situ hybridization. In contrastto earlier work that concluded that both NHE2 and NHE4 were expressedthroughout the intestine and in the kidney, our data show that there isno NHE2 message in the kidney and NHE4 is not expressed in small orlarge intestine. Analyses of intestinal epithelial and kidney membraneproteins by an NHE2-specific antibody identified a doublet at <90 kDain intestine but not in kidney. NHE2 is highly expressed in theNa+-absorptive epithelium ofjejunum, ileum, and ascending and descending colon. NHE4 mRNA messageis found in the inner medulla of the kidney as previously reported (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao,and E. B. Chang. J. Biol. Chem. 269:29704-29709, 1994) and not in the intestine. From these data, wespeculate that neither NHE2 nor NHE4 has a role in renalNa+ absorption. NHE2 is likelyinvolved in gut Na+ absorption,whereas NHE4 may have a specialized role in cell volume rectificationof inner medullary collecting duct cells. Knowledge of the correcttissue and cell-specific distribution of these two antiporters shouldhelp significantly in understanding their physiological roles. 相似文献
8.
M.Z. Abedin D.I.N. Giurgiu Z.R. Abedin E.A. Peck X. Su P.R. Smith 《The Journal of membrane biology》2001,182(2):123-134
Gallbladder Na+ absorption is linked to gallstone formation in prairie dogs. Na+/H+ exchange (NHE) is one of the major Na+ absorptive pathways in gallbladder. In this study, we measured gallbladder Na+/H+ exchange and characterized the NHE isoforms expressed in prairie dogs. Na+/H+ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na+ flux and apical 22Na+ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J
Na
ms was higher than J
Na
sm with J
Na
net absorption. Mucosal DMA inhibited transepithelial Na+ flux in a dose-dependent fashion, causing J
Na
ms equal to J
Na
sm and blocking J
Na
net absorption at 100 μm. Basal 22Na+ uptake rate was 10.9 ± 1.0 μmol · cm−2· hr−1 which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression
of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na+/H+ exchange accounts for a substantial fraction of gallbladder apical Na+ entry and most of net Na+ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na+ uptake and transepithelial Na+ absorption.
Received: 9 February 2001/Revised: 11 April 2001 相似文献
9.
Murtazina R Kovbasnjuk O Donowitz M Li X 《The Journal of biological chemistry》2006,281(26):17845-17855
A previous study showed that approximately 25-50% of rabbit ileal brush border (BB) Na(+)/H(+) exchanger NHE3 is in lipid rafts (LR) (Li, X., Galli, T., Leu, S., Wade, J. B., Weinman E. J., Leung, G., Cheong, A., Louvard, D., and Donowitz, M. (2001) J. Physiol. (Lond.) 537, 537-552). Here, we examined the role of LR in NHE3 transport activity using a simpler system: opossum kidney (OK) cells (a renal proximal tubule epithelial cell line) containing NHE3. approximately 50% of surface (biotinylated) NHE3 in OK cells distributed in LR by density gradient centrifugation. Disruption of LR with methyl-beta-cyclodextrin (MbetaCD) decreased NHE3 activity and increased K'(H+)(i), but K(m)((Na+)) was not affected. The MbetaCD effect was completely reversed by repletion of cholesterol, but not by an inactive analog of cholesterol (cholestane-3beta,5alpha,6beta-triol). The MbetaCD effect was specific for NHE3 activity because it did not alter Na(+)-dependent l-Ala uptake. MbetaCD did not alter OK cell BB topology and did not change the surface amount of NHE3, but greatly reduced the rate of NHE3 endocytosis. The effects of inhibiting phosphatidylinositol 3-kinase and of MbetaCD on NHE3 activity were not additive, indicating a common inhibitory mechanism. In contrast, 8-bromo-cAMP and MbetaCD inhibition of NHE3 was additive, indicating different mechanisms for inhibition of NHE3 activity. Approximately 50% of BB NHE3 and only approximately 11% of intracellular NHE3 in polarized OK cells were in LR. In summary, the BB pool of NHE3 in LR is functionally active because MbetaCD treatment decreased NHE3 basal activity. The LR pool is necessary for multiple kinetic aspects of normal NHE3 activity, including V(max) and K'(H+)(i), and also for multiple aspects of NHE3 trafficking, including at least basal endocytosis and phosphatidylinositol 3-kinase-dependent basal exocytosis. Because the C-terminal domain of NHE3 is necessary for its regulation and because the changes in NHE3 kinetics with MbetaCD resemble those with second messenger regulation of NHE3, these results suggest that the NHE3 C terminus may be involved in the MbetaCD sensitivity of NHE3. 相似文献
10.
11.
C. Bookstein M.W. Musch P.K. Dudeja R.L. McSwine Y. Xie T.A. Brasitus M.C. Rao E.B. Chang 《The Journal of membrane biology》1997,160(3):183-192
This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants
of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease
of ≈50% in the specific activities of both NHE1 and NHE3. Only V
max was affected; K
Na was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride,
5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely
unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues
in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular
transport by incubation at 4°C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect
does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity)
influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.
Received: 23 January 1997/Revised: 1 August 1997 相似文献
12.
Brian L. Lee Xiuju Li Yongsheng Liu Brian D. Sykes Larry Fliegel 《The Journal of biological chemistry》2009,284(17):11546-11556
The Na+/H+ exchanger isoform 1 is a ubiquitously
expressed integral membrane protein that regulates intracellular pH in mammals
by extruding an intracellular H+ in exchange for one extracellular
Na+. We characterized structural and functional aspects of the
critical transmembrane (TM) segment XI (residues 449-470) by using cysteine
scanning mutagenesis and high resolution NMR. Each residue of TM XI was
mutated to cysteine in the background of the cysteine-less protein and the
sensitivity to water-soluble sulfhydryl reactive compounds MTSET
((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES
((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues
with at least moderate activity remaining. Of the residues tested, only
proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The
activity of the L465C mutant was almost completely eliminated, whereas that of
the L457C and I461C mutants was partially affected. The structure of a peptide
representing TM XI (residues Lys447-Lys472) was
determined using high resolution NMR spectroscopy in dodecylphosphocholine
micelles. The structure consisted of helical regions between
Asp447-Tyr454 and Phe460-Lys471 at
the N and C termini of the peptide, respectively, connected by a region with
poorly defined, irregular structure consisting of residues
Gly455-Gly459. TM XI of NHE1 had a structural similarity
to TM XI of the Escherichia coli Na+/H+
exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with
residue Leu465 contributing to the pore.The mammalian Na+/H+ exchanger isoform 1
(NHE1)4 is a
ubiquitous integral membrane protein that regulates intracellular pH. It
mediates removal of a single intracellular proton in exchange for an
extracellular sodium ion (1).
NHE1 has many functions aside from protection of cells from intracellular
acidification (2). It promotes
cell growth and differentiation
(3), regulates sodium fluxes
and cell volume after challenge by osmotic shrinkage
(4), and has been demonstrated
to be involved in modulating cell motility
(5). In addition its activity
is important in invasiveness of neoplastic breast cancer cells
(6). NHE1 also plays critical
roles in heart disease. It has a contributing role in heart hypertrophy and in
the damage that occurs during ischemia and reperfusion. Inhibition of NHE1
with Na+/H+ exchanger inhibitors protects the myocardium
during various disease states
(7-10).NHE1 is composed of two general regions, an N-terminal membrane domain of
∼500 amino acids and a C-terminal regulatory domain of ∼315 amino
acids (1,
8). The membrane domain is
responsible for ion movement and an analysis of topology by cysteine scanning
accessibility suggested it has 3 membrane-associated segments and 12 integral
transmembrane segments (11)
(Fig. 1A). The
mechanism of transport of the membrane domain is of great interest both from a
scientific viewpoint and in the design of improved NHE1 inhibitors that may be
necessary for clinical use (1).
In this regard, we have recently characterized the functionally important
residues and the structure of both TM IV and TM VII. Prolines 167 and 168 of
TM IV were critical to NHE1 function
(12) and cysteine-scanning
mutagenesis was used to show that Phe161 is a pore lining residue
critical to transport. Analysis of the structure of TM IV showed that TM IV is
composed of one region of β-turns, an extended middle region including
Pro167-Pro168, and a helical region
(13). TM VII was much more
typical of a transmembrane helix although it was interrupted with a break in
the helix at the functionally critical residues
Gly261-Glu262
(14).Open in a separate windowFIGURE 1.Models of the Na+/H+ exchanger.
A, simplified topological model of the transmembrane domain of the
NHE1 isoform of the Na+/H+ exchanger as described
earlier (11). EL,
extracellular loop; IL, intracellular loop. B, model of amino acids
present in TM XI.Another important TM segment of the Na+/H+ exchanger
is TM XI (Fig. 1B).
Several different lines of evidence have suggested that it is critical to NHE1
function. A recent study generated chimeras of NHE1 from various species and
found that a region including TM XI was important in determining NHE1
inhibitor sensitivity (15).
More specifically, mutagenesis of several amino acids of TM XI has shown that
it is likely involved in either ion transport or proper targeting to the
plasma membrane. Two mutants in TM XI, Y454C and R458C, are retained in the
endoplasmic reticulum (16). In
addition, mutation of Gly455 and Gly456 in TM XI shift
the pHi dependence of the exchanger to the alkaline side,
whereas mutation of Arg440 in intracellular loop 5 at the
N-terminal end of TM XI shifts the pHi dependence to make
it more acidic (17,
18). Also, the structure of
the bacterial Na+/H+ exchanger NhaA has been elucidated.
Both TM IV and TM XI play a critical role forming an assembly that cross, with
each being a helix, an extended polypeptide and a short helix
(19). We found that TM IV of
NHE1 has a similar structure and function to that of TM IV of NhaA
(2,
13), leaving open the
possibility that TM XI of NHE1 is also similar in structure and function to TM
XI of NhaA.For these reasons, we undertook a systematic examination of the structural
and functional aspects of TM XI of the NHE1 isoform of the
Na+/H+ exchanger. The sequence of human TM XI of NHE1 is
449QFIIAYGGLRGAIAFSLGYLLD470. In this study we use
cysteine scanning mutagenesis and site-specific mutagenesis to identify and
characterize critical pore lining residues of the protein. We also use nuclear
magnetic resonance (NMR) spectroscopy to characterize the structure of a
synthetic peptide representing TM XI in dodecylphosphocholine (DPC) micelles.
Evidence has suggested that TM segments of membrane proteins possess all the
structural information required to form their higher order structures in their
amino acid sequence (20). This
has been demonstrated in earlier studies on membrane protein segments such as
the cystic fibrosis transmembrane conductance regulator
(21), a fungal
G-protein-coupled receptor
(22), bacteriorhodopsin
(23,
24), and rhodopsin
(25), where it was shown that
isolated TM segments from membrane proteins had structures in good agreement
with the segments of the entire protein. Also, the use of DPC micelles has
been shown to be an excellent membrane mimetic environment for these studies
(26,
27). Our study identifies
Leu465 as contributing to the pore of the protein and shows that
the structure of TM XI consists of two helices corresponding to
Asp447-Tyr454 and Phe460-Lys471 at
the N and C termini, respectively, connected by a flexible region at residues
455-459. The structure of TM XI was similar to the x-ray structure of TM XI of
NhaA. 相似文献
13.
Na+/H+ exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells 下载免费PDF全文
Anja Paehler vor der Nolte Giriprakash Chodisetti Zhenglin Yuan Florian Busch Brigitte Riederer Min Luo Yan Yu Manoj B. Menon Andreas Schneider Renata Stripecke Katerina Nikolovska Sunil Yeruva Ursula Seidler 《Journal of cellular physiology》2017,232(7):1669-1680
Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+/H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1‐dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1‐dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ~10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady‐state pHi and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process. 相似文献
14.
Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe 总被引:3,自引:0,他引:3
McSwine Rebecca L.; Musch Mark W.; Bookstein Crescence; Xie Yue; Rao Mrinalini; Chang Eugene B. 《American journal of physiology. Cell physiology》1998,275(3):C693
We examined the regulation of theNa+/H+exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinalC2/bbe cells, which spontaneously differentiate and have little basalapical NHE activity. Unidirectional apical membrane22Na+influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of5-(N,N-dimethyl)amiloride-inhibitableflux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHEactivity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulatedby serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3exhibited similar patterns of regulation, suggesting that the secondmessenger effects observed were not secondary to effects on cell pH.Thus, in contrast to their expression in nonepithelial cells, NHE2 andNHE3 expressed in an epithelial cell line behave similarly toendogenously expressed intestinal apical membrane NHEs. We concludethat physiological regulation and function of epithelium-specific NHEsare dependent on tissue-specific factors and/or conditionalrequirements. 相似文献
15.
C. Bookstein M.W. Musch Y. Xie M.C. Rao E.B. Chang 《The Journal of membrane biology》1999,171(1):87-95
Until recently, studies to characterize the intestinal epithelial Na+/H+ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na+/H+ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure
NHE activity. Because changes in pH
i
only approximate Na+/H+ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation,
we have used 22[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these
measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in
the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated
by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition
by cAMP and Ca++ involves changes to both K
Na
and V
max
. In contrast, the same two second messengers inhibit NHE3 by a decrease in V
max
exclusively. Phorbol ester activation of protein kinase C alters both V
max
and K
Na
of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active
and are differentially regulated by signal transduction pathways.
Received: 28 January 1999/Revised: 18 May 1999 相似文献
16.
Köster S Pavkov-Keller T Kühlbrandt W Yildiz Ö 《The Journal of biological chemistry》2011,286(47):40954-40961
The ubiquitous mammalian Na+/H+ exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca2+-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1CaMBR) in complex with CaM and Ca2+. The C- and N-lobes of CaM bind the first and second helix of NHE1CaMBR, respectively. Both the NHE1 helices and the Ca2+-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca2+ regulates NHE1 activity. 相似文献
17.
The Na(+)/H(+) exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H(+) concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na(+) or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range. 相似文献
18.
Abu Jawdeh BG Khan S Deschênes I Hoshi M Goel M Lock JT Shinlapawittayatorn K Babcock G Lakhe-Reddy S DeCaro G Yadav SP Mohan ML Naga Prasad SV Schilling WP Ficker E Schelling JR 《The Journal of biological chemistry》2011,286(49):42435-42445
Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2). 相似文献
19.
Collazo R Fan L Hu MC Zhao H Wiederkehr MR Moe OW 《The Journal of biological chemistry》2000,275(41):31601-31608
Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule Na(+) transport via its action on the apical membrane Na(+)/H(+) exchanger NHE3. In the opossum kidney cell line, inhibition of NHE3 activity was detected from 5 to 45 min after PTH addition. Increase in NHE3 phosphorylation on multiple serines was evident after 5 min of PTH, but decrease in surface NHE3 antigen was not detectable until after 30 min of PTH. The decrease in surface NHE3 antigen was due to increased NHE3 endocytosis. When endocytic trafficking was arrested with a dominant negative dynamin mutant (K44A), the early inhibition (5 min) of NHE3 activity by PTH was not affected, whereas the late inhibition (30 min) and decreased surface NHE3 antigen induced by PTH were abrogated. We conclude that PTH acutely inhibits NHE3 activity in a biphasic fashion by NHE3 phosphorylation followed by dynamin-dependent endocytosis. 相似文献
20.
Graham H. Diering Yuka Numata Steven Fan John Church Masayuki Numata 《Molecular biology of the cell》2013,24(21):3435-3448
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na+/H+ exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase–Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation. 相似文献