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R R Kopito

Publications and source records attributed to R R Kopito.

At least 37 records · Page 2Linked to original sources
PubMed1996

Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation.

The common delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe508 deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and delta F508 in vivo. Incubation of transient and stable delta F508 transfectants with 10% glycerol induced a significant accumulation of delta F508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued delta F508 polypeptides form functional plasma membrane CFTR channels. These effects were dose- and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) delta F508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.

Cells, Cultured↗
PubMed1996

Immunocytochemical localization of vacuolar H+-ATPase and Cl--HCO3- anion exchanger (erythrocyte band-3 protein) in avian osteoclasts: effect of calcium-deficient diet on polar expression of the H+-ATPase pump.

Osteoclasts attach to the bone surface and resorb bone by secreting protons into an isolated subosteoclastic compartment. Previous studies have shown the presence of a vacuolar type H+-ATPase, and a functional Cl--HCO3- anion exchanger in the osteoclast. In the present studies, using a monoclonal antibody to the 31-kDa subunit of H+-ATPase and a rabbit antiserum to the erythrocyte band-3 protein (Cl--HCO3- anion exchanger) we have immunocytochemically localized the respective pumps in bone sections obtained from chickens fed a normal or a calcium-deficient diet for 4 weeks. Our results indicate that although H+-ATPase is either evenly distributed throughout the osteoclast or is more polarized at its ruffled membrane juxtaposed to the bone surface, the band-3 protein immunoreactivity is always localized to the plasma membrane which is not attached to the bone surface (basolateral membrane). Four weeks of a calcium-deficient diet resulted in a significant increase in the percentage of osteoclasts that were polarized for the H+-ATPase pump at their ruffled membrane, and a trend toward increased total number of osteoclasts, although the latter did not reach statistical significance (P = 0.09). These changes were not accompanied by a significant increase in the intensity of staining for H+-ATPase. Band-3 protein immunoreactivity was always prominent, limited to the basolateral membrane, and did not alter with calcium-deficient diet or with changes in the degree of H+-ATPase polarization.

Adenosine Triphosphatases↗
PubMed1995

A conserved glutamate is responsible for ion selectivity and pH dependence of the mammalian anion exchangers AE1 and AE2.

The erythrocyte anion exchanger AE1 (band 3) serves as an important model for the study of the mechanism of ion transport. Chemical modification of human erythrocyte AE1 has previously suggested that glutamic acid residue 681 lies within the transport pathway and can cross the permeability barrier. This glutamate is conserved in all anion exchangers sequenced to date. We examined the effect on divalent (sulfate) and monovalent (chloride and bicarbonate) anion transport of mutating the corresponding glutamates in mouse AE1 and the closely related anion exchanger, AE2. Substitution of this conserved glutamate with uncharged or basic amino acids had a negligible effect on the maximal rate of sulfate-sulfate exchange in AE-reconstituted proteoliposomes, but largely abolished the steep pH dependence of sulfate transport observed in wild-type AE1 and AE2. In contrast, exchange of monovalent anions was undetectable in cells expressing these mutants. Replacement of the conserved glutamate with aspartate abolished both monovalent and divalent anion transport. These data suggest that the conserved glutamate residue plays a dual role in determining anion selectivity and in proton coupling to sulfate transport. A model explaining the role of the conserved glutamate in promoting ion selectivity and pH regulation is discussed.

Animals↗
PubMed1995

Degradation of CFTR by the ubiquitin-proteasome pathway.

Most cases of cystic fibrosis are caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to the rapid degradation of CFTR molecules that have not matured beyond the endoplasmic reticulum (ER). The mechanism by which integral membrane proteins including CFTR are recognized and targeted for ER degradation and the proteolytic machinery involved in this process are not well understood. We show here that the degradation of both wild-type and mutant CFTR is inhibited by two potent proteasome inhibitors that induce the accumulation of polyubiquitinated forms of immature CFTR. CFTR degradation was also inhibited by coexpression of a dominant negative ubiquitin mutant and in cells bearing a temperature-sensitive mutation in the ubiquitin-activating enzyme, confirming that ubiquitination is required for rapid CFTR degradation.

Cysteine Endopeptidases↗
PubMed1995

Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis.

CFTR is a member of the traffic ATPase superfamily and a Cl- ion channel that appears to require ATP hydrolysis for gating. Analysis of single CFTR Cl- channels reconstituted into planar lipid bilayers revealed the presence of two open conductance states that are connected to each other and to the closed state by an asymmetric cycle of gating events. We show here that the transition between the two open conductance states is directly coupled to ATP hydrolysis by one of the consensus nucleotide-binding folds, designated NBF2. Moreover, the transition between the closed state and one of the open states is linked to the binding of ATP. This analysis permits real-time visualization of conformational changes associated with a single cycle of ATP hydrolysis by a single protein molecule and suggests a model describing a role for ATP in CFTR gating.

Adenosine Triphosphate↗
PubMed1995

Sulfate transport mediated by the mammalian anion exchangers in reconstituted proteoliposomes.

The kinetic properties of sulfate transport mediated by the anion exchangers AE1 and AE2 have been examined. Microsomes isolated from HEK cells transiently overexpressing either protein were reconstituted in unilamellar, 200-600-nm diameter proteoliposomes. Transport mediated by the exchangers was monitored by loading the reconstituted proteoliposomes with the slowly transportable anion SO4(2-) using [35S]SO4(2-) as a tracer and performing [35S]SO4(2-)/SO4(2-) exchange. The following data suggest that AE1 and AE2 have been functionally reconstituted: (i) the rate of SO4(2-) transport in AE1 and AE2 containing proteoliposomes was 10-20 times higher than in proteoliposomes derived from control microsomes; (ii) the transport of SO4(2-) was strongly dependent on the presence of a trans anion; and (iii) the anion exchanger inhibitors, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2,2'-di-sulfonate (DIDS) totally abolished SO4(2-) transport. furthermore, DIDS inhibits SO4(2-) transport only when occluded inside the vesicles, indicating a uniform, asymmetrical, inside-out orientation of the reconstituted exchangers. The Ki values of the stilbene disulfonate compound DNDS were 2.5 and 4 microM for AE1 and AE2, respectively, suggesting that the two exchangers possess similar high affinity sites for stilbene compounds. Both AE1 and AE2 showed the same steep pH dependence of sulfate transport, which was maximal at pH 5.5 and reduced to less than 10% (of the value at pH 5.5) at pH 8.5, suggesting that an acidic residue shared by AE1 and AE2 participates in the pH regulation of sulfate transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

The role of cysteine residues in the erythrocyte plasma membrane anion exchange protein, AE1.

AE1 (Band 3), a congruent to 110-kDa integral plasma membrane protein, facilitates the electroneutral movement of Cl- and HCO3- across the erythrocyte membrane and serves as the primary attachment site for the erythrocyte spectrin-actin cytoskeleton. In this investigation, we have characterized the role of native cysteines in the function of AE1. We have constructed a mutant version of human AE1 (AE1C-) in which all five cysteines of AE1 were replaced with serines. Wild-type and AE1C- cDNAs were expressed by transient transfection of human embryonic kidney cells. Two of the mutated cysteines in AE1C- are in a region involved in ankyrin binding, and ankyrin binding has previously been shown to be sensitive to the oxidation state of these cysteines. However, the KD values for ankyrin binding by AE1 and AE1C- were indistinguishable, suggesting that AE1 cysteines are not essential components of the ankyrin-binding site. Using size exclusion chromatography, both AE1 and AE1C- were found to associate as a mixture of dimers and high molecular mass complexes. The rate of anion exchange by AE1C-, as measured in a reconstituted microsome sulfate transport assay, was indistinguishable from that by AE1 and was inhibited by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate. We conclude that the cysteines of AE1 are not required for the anion exchange or cytoskeletal binding roles of the protein.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1994

The major kidney AE1 isoform does not bind ankyrin (Ank1) in vitro. An essential role for the 79 NH2-terminal amino acid residues of band 3.

The AE1 (band 3) protein mediates the exchange of anions across the erythrocyte plasma membrane and, via association with the adapter molecule, ankyrin (Ank1), forms the major link between the membrane and the underlying spectrin cytoskeleton. The major kidney isoform of AE1 (kAE1), a protein that is otherwise identical to erythroid AE1 but lacks the NH2-terminal 79 amino acids, is localized to the basolateral plasma membrane of acid-secreting (alpha-type) intercalated cells of distal nephron. It has been proposed that this polarized distribution of kAE1 is due, at least in part, to its association with the ankyrin-spectrin cytoskeleton. In this study, we have used cell-free binding assays to investigate the interaction of anion exchangers with an Ank1 fragment, R13-H, that contains the AE1 binding site. Microsomes from cells expressing full-length erythroid AE1 bound 125I-labeled R13-H, revealing the presence of both high (Kd = 12.5 nM) and low (Kd = 166 nM) affinity sites. This binding was specific, as evidenced by the failure to observe high affinity binding of 125I-R13-H to microsomes from cells transfected with vector alone or with AE1m, a mutant lacking the approximately 400 amino acid NH2-terminal cytoplasmic ankyrin binding domain. Using this assay, we could detect no high affinity association between kAE1 and 125I-R13-H. We conclude that the NH2-terminal 79 amino acids play an essential role in high affinity and specific binding of AE1 to Ank1.

Amino Acids↗
PubMed1994

Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins.

Mutant (delta F508) and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) were synthesized initially as an approximately 140-kDa core-glycosylated precursor, which, in the case of wild-type CFTR, was chased to an approximately 160 kDa form bearing complex oligosaccharides. Mutant CFTR disappeared from the detergent-soluble cell extract with rapid (t1/2 = 27 min) kinetics. Only approximately 25% of the initially synthesized wild-type 140-kDa CFTR precursor was detected as mature protein; the remaining approximately 75% decayed with kinetics (t1/2 = 33 min) indistinguishable from those of the mutant. Rapid degradation kinetics and inefficient processing of wild-type CFTR were also observed in the colonic carcinoma lines HT29 and T84 and in stably transfected C127 cells, which express 5-50 times lower levels of CFTR. These results suggest that inefficient processing and rapid degradation of wild-type CFTR precursor are an intrinsic property of CFTR in these diverse cell types and are not an artifact of overexpression. Degradation of wild-type and mutant 140-kDa CFTR began without significant lag following synthesis. These data suggest that wild-type and delta F508 CFTR differ in the efficiency of folding of the core-glycosylated primary translation product.

Amino Acid Sequence↗
PubMed1994

Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating.

Single channel analysis of artificial lipid planar bilayers reconstituted with wild-type human cystic fibrosis transmembrane regulator (CFTR) revealed a 10.3 pS Cl- selective channel that was activated upon phosphorylation with protein kinase A. Gating of this channel was described by a simple kinetic model consisting of a single open burst state and two closed states. The open probability of CFTR channels in bilayers increased as a function of increasing Mg-ATP concentration and exhibited negative cooperativity, suggesting the interaction of two or more ATP binding sites in channel gating. Mg-ATP increased channel open probability by decreasing the duration of the long-lived closed state but had no effect on either the mean open time or the fast closed state. ADP inhibited channel opening by precisely antagonizing the effect of ATP, suggesting that ADP inhibits the CFTR channel by competing with ATP for binding. Poorly hydrolyzable ATP analogs such as AMP-PNP and ATP gamma S, polyphosphates such as pyrophosphate (PPi) and tripolyphosphate (PPPi), and orthovanadate failed to support channel activity alone. When applied in the presence of ATP, these compounds all caused the CFTR channel to "lock" into a prolonged open burst state. These data support a model in which hydrolysis of ATP leads to closure of channels that have been opened by ATP.

Adenosine Monophosphate↗
PubMed1994

AE3 anion exchanger isoforms in the vertebrate retina: developmental regulation and differential expression in neurons and glia.

Plasma membrane anion exchangers constitute a multigene family that contributes to the regulation of intracellular pH and chloride concentration in many cell types. We have characterized two polypeptide isoforms of the AE3 gene that are expressed in the rat retina. Using antipeptide antibodies specific for defined NH2-terminal and COOH-terminal epitopes, we have identified a 165 kDa polypeptide whose expression is restricted to the primary glial cell type of the retina, the Müller cell, and a 125 kDa polypeptide that is expressed in horizontal neurons. Expression of the Müller cell isoform exhibits a polarized distribution and is highest in basal endfoot processes. These AE3 isoforms exhibit a distinct developmental expression pattern in postnatal rat retina. The neuronal isoform is undetectable in neonatal retina until postnatal day 10-15, correlating strongly with the onset of retinal function.

Aging↗
PubMed1993

Cl-/HCO3- exchange function differs in adult and fetal rat hippocampal neurons.

We studied Cl-/HCO3- exchange function in acutely dissociated single hippocampal neurons from adult and fetal day 18 rat using a fluorescent intracellular pH (pHi) indicator dye. The presence of Cl-/HCO3- exchange activity was assayed by observing the elevation in pHi upon acute reversal of the Cl- gradient. Resting intracellular pH in acutely dissociated neurons of both adult and fetal tissue was significantly higher than that of cultured fetal hippocampal neurons (day 10-12 in culture). Acute removal of extracellular Cl- caused a rapid and reversible increase in pHi by 0.25 pH units in adult neurons but had virtually no effect in similarly dissociated fetal neurons. Cl-/HCO3- exchange activity was also undetectable in fetal cultured hippocampal neurons. The mRNA for the anion exchanger AE3 is expressed abundantly in adult rodent neurons. AE3 is a potential candidate molecule for the observed Cl-/HCO3- exchange activity. In situ hybridization was used to monitor expression of the AE3 gene in these two age groups. We found that both adult and fetal neurons express AE3 mRNA. These results indicate that AE3 may not function as a Cl-/HCO3- exchanger in fetal neurons, in contrast to its possible role in the adult brain.

Acid-Base Equilibrium↗
PubMed1993

Functional activation of plasma membrane anion exchangers occurs in a pre-Golgi compartment.

Folding and oligomerization of most plasma membrane glycoproteins, including those involved in ion transport, occur in the ER and are frequently required for their exit from this organelle. It is currently unknown, however, where or when in the biosynthetic pathway these proteins become functionally active. AE1 and AE2 are tissue-specific, plasma membrane anion transport proteins. Transient expression of AE2 in a eukaryotic cell line leads to an increase in stilbene inhibitable whole cell 35SO4(2-)-efflux consistent with its function as a plasma membrane anion exchanger. No such increased transport activity was observed in AE1 transfectants, despite the fact that the two proteins were synthesized in roughly equal portions. In contrast, both AE1 and AE2 expression resulted in significant increase in Cl-/SO4(2-)-exchange in crude microsomes demonstrating that both AE1 and AE2 cDNAs encode functional proteins. Immunofluorescence staining and pulse-chase labeling experiments revealed that while 60% of AE2 is processed to the cell surface of transfectants, AE1 is restricted to an intracellular compartment and never acquires mature oligosaccharides. Crude microsomes from transfected cells were fractionated into plasma membrane and ER-derived vesicles by con A affinity chromatography. All of the AE1 and approximately half of the cellular AE2 was eluted with the ER vesicles, confirming their intracellular localization. Anion transport measurements on these fractions confirmed that the ER-restricted anion exchangers were functional. We conclude that AE1 and AE2 acquire the ability to mediate anion exchange at an early stage of their biosynthesis, before their exit from the ER.

Animals↗
PubMed1993

Association of the brain anion exchanger, AE3, with the repeat domain of ankyrin.

The 89 kDa NH2-terminal domain of erythrocyte ankyrin is composed almost entirely of 22 tandem repeats of a 33 amino acid sequence and constitutes the binding site for the cytoplasmic NH2-terminal domain of the erythrocyte anion exchanger, AE1. We have developed an assay to evaluate the in vivo interaction between a fragment of ankyrin corresponding to this domain (ANK90) and two non-erythroid anion exchangers, AE2 and AE3, that share considerable structural homology with AE1. Association was assessed by co-immunoprecipitation of ANK90-anion exchanger complexes from detergent extracts of cells cotransfected with plasmids encoding the ankyrin fragment and the anion exchanger or mutants thereof. ANK90 was co-immunoprecipitated with AE1 but not with an AE1 deletion mutant lacking the cytoplasmic NH2-terminal domain. Using this assay, we show that the brain anion exchanger AE3, but not the closely related homologue, AE2, is capable of binding to ankyrin.

Anion Transport Proteins↗
PubMed1993

Generation of truncated brain AE3 isoforms by alternate mRNA processing.

AE3 gene is a member of the AE anion exchanger gene family that is expressed primarily in brain and heart. The principal product of the AE3 gene in rodent brain, FL-AE3p, when expressed in heterologous cell lines, gives rise to chloride-dependent changes in intracellular pH consistent with its operation as an anion exchanger. We have identified two novel isoforms of mouse AE3 that are generated by tissue-specific alternate RNA processing. One of these isoforms encodes a polypeptide, 14-AE3p, that corresponds to a portion of the NH2-terminal cytoplasmic domain of AE3. 14-AE3p lacks the entire transmembrane domain that-in FL-AE3p forms the anion exchange channel. Immunoblots with antibodies to the NH2- and COOH-termini confirm that FL-AE3 and 14-AE3 are expressed in rat brain as 160 kDa and 74 kDa polypeptides, respectively. Unlike FL-AE3p, however, 14-AE3p is insoluble in non-ionic detergent, suggesting a possible association with the cytoskeleton.

Alternative Splicing↗
PubMed1991

Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line.

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Cystic fibrosis gene expression is not correlated with rectifying Cl- channels.

Cystic fibrosis (CF) involves a profound reduction of Cl- permeability in several exocrine tissues. A distinctive, outwardly rectifying, depolarization-induced Cl- channel (ORDIC channel) has been proposed to account for the Cl- conductance that is defective in CF. The recently identified CF gene is predicted to code for a 1480-amino acid integral membrane protein termed the CF transmembrane conductance regulator (CFTR). The CFTR shares sequence similarity with a superfamily of ATP-binding membrane transport proteins such as P-glycoprotein and STE6, but it also has features consistent with an ion channel function. It has been proposed that the CFTR might be an ORDIC channel. To determine if CFTR and ORDIC channel expression are correlated, we surveyed various cell lines for natural variation in CFTR and ORDIC channel expression. In four human epithelial cell lines (T84, CaCo2, PANC-1, and 9HTEo-/S) that encompass the full observed range of CFTR mRNA levels and ORDIC channel density we found no correlation.

Base Sequence↗