Biomedical subjects

S L Alper

Publications and source records attributed to S L Alper.

At least 55 records · Page 3Linked to original sources

PubMed1999

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation.

Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of (86)Rb into Xenopus oocytes requires extracellular Cl(-), is blocked by the diuretic R(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

3' Untranslated Regions↗

PubMed1999

Expression of AE2 anion exchanger in mouse intestine.

We have characterized expression of anion exchanger 2 (AE2) mRNA and protein in the mouse intestine. AE2 mRNA abundance was higher in colon than in more proximal segments. AE2a mRNA was more abundant than AE2b mRNA throughout the intestine, and AE2c mRNA was expressed at very low levels. This AE2 mRNA pattern contrasted with that in mouse stomach, in which AE2c > AE2b > AE2a. AE2 polypeptide abundance as detected by immunoblot qualitatively paralleled that of mRNA, whereas AE2 immunostaining exhibited a more continuous decrease in intensity from colon to duodenum. AE2 polypeptide was more abundant in colonic surface cells than in crypts, whereas ileal crypts and villi exhibited similar AE2 abundance. AE2 was also observed in mural and vascular smooth muscle. Localization of AE2 epitopes was restricted to the basolateral membranes of epithelial cells throughout the intestine with three exceptions. Under mild fixation conditions, anti-AE2 amino acids (aa) 109-122 detected nonpolarized immunostaining of ileal enterocytes and of Paneth cell granule membranes. An epitope detected by anti-AE2 aa 1224-1237 was also localized to subapical regions of Brunner's gland ducts of duodenum and upper jejunum. These localization studies will aid in the interpretation of anion exchanger function measured in epithelial sheets, isolated cells, and membrane vesicles.

Animals↗

PubMed1999

Immunolocalization of anion exchanger AE2 and Na(+)-HCO(-)(3) cotransporter in rat parotid and submandibular glands.

Salivary glands secrete K(+) and HCO(-)(3) and reabsorb Na(+) and Cl(-), but the identity of transporters involved in HCO(-)(3) transport remains unclear. We investigated localization of Cl(-)/HCO(-)(3) exchanger isoform AE2 and of Na(+)-HCO(-)(3) cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at approximately 160 and 180 kDa for AE2 and approximately 130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of HCO(-)(3) by rat salivary duct cells than previously believed.

Animals↗

PubMed1999

Distribution in rat brain of binding sites of kaliotoxin, a blocker of Kv1.1 and Kv1.3 alpha-subunits.

The distribution of the binding sites for kaliotoxin (KTX), a blocker of voltage-dependent K(+) channels, was studied with quantitative autoradiography in adult rat brain and during postnatal brain maturation. Iodinated KTX bound specifically to tissue sections with a high affinity (K(d) = 82 pM) and a maximal binding capacity of 13.4 fmol/mg protein. The distribution of KTX binding sites within the central nervous system was heterogeneous. The highest densities were found in the neocortex, hypothalamus, dentate gyrus, bed nucleus of the stria terminalis, and parabrachial nuclei. The lowest level was observed in the white matter. From postnatal day 5 onward, KTX binding sites were detectable only in the hindbrain. The density of KTX binding sites in whole brain drastically increased after postnatal day 15 to achieve adult levels at postnatal day 60 in the whole brain. Bath application of KTX to Xenopus laevis oocytes blocked recombinant Kv1.3 and Kv1.1 channels potently and Kv1.2 channels less potently, with respective K(d) values of 0.1, 1.5, and 25 nM. KTX affinities for each of these channels expressed in mammalian cells were about 10-fold lower. A comparison of the distribution of KTX binding sites with that of Kv1 channel polypeptides, together with the pharmacology of KTX block, suggests that the principal targets for KTX in rat brain are K(+) channels containing Kv1.1 and Kv1.3 alpha-subunits.

Aging↗

PubMed1998

Novel AE1 mutations in recessive distal renal tubular acidosis. Loss-of-function is rescued by glycophorin A.

The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport.

Acidosis, Renal Tubular↗

PubMed1998

The guinea pig cochlear AE2 anion exchanger: cDNA cloning and in situ localization within the cochlea.

This study has characterized the repertoire of the anion exchanger (AE) family members expressed within the guinea pig organ of Corti, the auditory neuroepithelia. Both AE2 and AE3 cDNAs were present, but AE1 cDNA was not detected. The more abundant AE2 was sequenced and its expression characterized in the cochlea. The 3888 base pairs (bp) AE2 sequence, compiled from multiple clones, includes 150 bp of upstream non-coding sequence and 3717 bp of open reading frame encoding a protein of 1238 amino acids. Immunoblot of cochlear homogenate revealed a single AE2-immunoreactive band of Mr 180 kDa. In situ hybridization and immunohistochemical analysis localized AE2 expression to several tissues and cell types within the guinea pig inner ear, including superior half of the spiral ligament and within the interdental cells lining the spiral limbus. However, AE2 was not clearly detected in the outer hair cells (OHC) of the organ of Corti by either immunohistochemistry or in situ hybridization. The results of these studies imply a physiologic role of AE2 in the cochlear homeostasis, but do not support its role as a potential 'motor protein' in mediating the in vitro-observed voltage-gated, ATP-independent OHC motility.

Amino Acid Sequence↗

PubMed1998

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

Animals↗

PubMed1998

Autosomal dominant distal renal tubular acidosis is associated in three families with heterozygosity for the R589H mutation in the AE1 (band 3) Cl-/HCO3- exchanger.

Distal renal tubular acidosis (dRTA) is characterized by defective urinary acidification by the distal nephron. Cl-/HCO3- exchange mediated by the AE1 anion exchanger in the basolateral membrane of type A intercalated cells is thought to be an essential component of lumenal H+ secretion by collecting duct intercalated cells. We evaluated the AE1 gene as a possible candidate gene for familial dRTA. We found in three unrelated families with autosomal dominant dRTA that all clinically affected individuals were heterozygous for a single missense mutation encoding the mutant AE1 polypeptide R589H. Patient red cells showed approximately 20% reduction in sulfate influx of normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitivity and pH dependence. Recombinant kidney AE1 R589H expressed in Xenopus oocytes showed 20-50% reduction in Cl-/Cl- and Cl-/HCO3- exchange, but did not display a dominant negative phenotype for anion transport when coexpressed with wild-type AE1. One apparently unaffected individual for whom acid-loading data were unavailable also was heterozygous for the mutation. Thus, in contrast to previously described heterozygous loss-of-function mutations in AE1 associated with red cell abnormalities and apparently normal renal acidification, the heterozygous hypomorphic AE1 mutation R589H is associated with dominant dRTA and normal red cells.

Acidosis, Renal Tubular↗

PubMed1998

Clotrimazole is a potent vasodilator of the rat coronary microcirculation.

Clotrimazole (CLT), used in the treatment of patients with sickle cell disease, directly blocks Ca2+-activated K+ (K+ Ca) channels in red cells and in portal vein smooth muscle cells by a cytochrome P450(cyt P450)-independent mechanism. Therefore, we examined the effects of CLT on vasomotor tone of coronary arterioles. Rat coronary arterioles (80-180 micro(m) in diameter) were studied in vitro in a pressurized no-flow state with a video microscopy. CLT (0.1 micromol/L) elicited in nonprecontracted vessels a small contraction (<10% baseline diameter, P < 0.05 vs time control), consistent with blockade of a hyperpolarizing K+ channel. However, similar contraction was produced by the cyt P450 blocker 17-octadecynoic acid (17-ODYA, 100 micromol/L), suggesting possible involvement of arachidonate metabolites of cyt P450. In contrast, microvessels precontracted with the thromboxane A2 analog U46619 dilated in response to CLT [>90% relaxation of the U46619-induced precontraction at 100 micromol/L (P < 0.01 vs time control)] and its structural analogs flutrimazole (FLT), UR-4055, UR4057, UR-4058, and UR-4059. This relaxation was cyt P450-independent, since the in vivo CLT metabolite (CLT-carbinol) was equipotent with CLT, and 17-ODYA did not promote relaxation. CLT-induced dilation was not inhibited by the nitric oxide synthase inhibitor NGnitro-l-arginine (100 micromol/L, P > 0.5) or affected by endothelial denudation (P > 0.5). Thus, CLT at concentrations >1 micromol/L is a potent vasodilator of rat coronary arterioles. This dilation is likely mediated through a vascular smooth muscle mechanism independent of cyt P450 and is not modulated by nitric oxide or by the endothelium. This effect may arise from CLT's reported ability to inhibit voltage-gated Ca2+ channels or to inhibit, in some tissues, Ca2+ release from intracellular stores. The CLT- and FLT-induced relaxation may be a property common to this class of drugs and have clinical applicability.

Animals↗

PubMed1998

Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic squalamine.

Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 microM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 microM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.

Animals↗

PubMed1998

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells.

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl- secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl- conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I- > Cl-; for forskolin, Cl- > I-). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I- > Cl-). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.

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

PubMed1998

Functional and molecular characterization of luminal and basolateral Cl-/HCO-3 exchangers of rat thick limbs.

Cl-/HCO-3 exchange was measured in luminal (LMV) and basolateral (BLMV) membrane vesicles purified from rat medullary thick ascending limb (MTAL). Cl-/HCO-3 exchange in BLMV and LMV was inhibited by DIDS, with respective IC50 values of 3.2 +/- 0.9 and 15.2 +/- 5.2 microM, whereas Cl- conductances were DIDS insensitive. At constant external pH, BLMV 36Cl-/HCO-3 and 36Cl-/Cl- exchanges exhibited a sigmoidal pattern of activation as internal pH (pHi) increased from 6.1 to 8.0, whereas LMV 36Cl-/Cl- exchange was unchanged between pHi 6.7 and 7.8. The 165-kDa AE2 polypeptide and approximately 115-kDa AE1-related polypeptide were present only in BLMV. In contrast, AE1-related polypeptides of approximately 90 and 95 kDa were present not only in BLMV but also (in variable abundance) in LMV. We conclude that rat MTAL BLMV and LMV express distinct anion exchange activities and distinct sets of AE polypeptides. AE2 (and perhaps AE1) in BLMV likely contribute to HCO-3 absorption. In contrast, LMV exchangers may contribute to NaCl absorption via parallel coupling with the luminal Na+/H+ antiporters and/or may provide negative feedback regulation of HCO-3 absorption.

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

PubMed1998

Mannitol at clinical concentrations activates multiple signaling pathways and induces apoptosis in endothelial cells.

BACKGROUND AND PURPOSE: Hyperosmotic mannitol therapy is widely used in the clinical setting for acute and subacute reduction in brain edema, to decrease muscle damage in compartment syndrome, and to improve renal perfusion. Though beneficial rheological effects commonly are attributed to mannitol, its direct effects on endothelial cells are poorly understood. METHODS: We studied the effect of hypertonic and hypotonic stress on bovine aortic endothelial (BAE) cells, using mannitol, urea, and sodium chloride and medium dilution in vitro. RESULTS: Exposure to incremental osmolar concentrations of 300 mOsm of each osmotic agent increased apoptosis in BAE cells (mannitol congruent withNaCl>urea). Induced programmed cell death was detected by DAPI staining of intact cell nuclei, and by TUNEL and DNA fragmentation ladder assays. Mannitol-induced apoptosis exhibited dose dependence (42% of cells at 300 mOsm [P<0.0001] compared with 1.2% of control cells) and was also observed in bovine smooth muscle cells. Mannitol-induced apoptosis was attenuated approximately 50% in the presence of cycloheximide or actinomycin D. Hypertonic mannitol and NaCl, but not urea, increased tyrosine phosphorylation of the focal adhesion contact-associated proteins paxillin and FAK. Hypotonic medium, which did not lead to apoptosis, increased protein tyrosine phosphorylation of FAK but not of paxillin. Addition of mannitol or NaCl also produced sustained increases in c-Jun NH2-terminal kinase (JNK) activity. In addition, hypertonic mannitol increased intracellular free [Ca2+] in a dose-dependent manner. Chelation of intracellular Ca2+ with quin2-AM (10 micromol/L) inhibited mannitol-induced apoptosis approximately 50%, as to a lesser extent did inhibition of tyrosine kinase activity with herbimycin (1 micromol/L). CONCLUSIONS: We have shown that hypertonic mannitol exposure induces endothelial cell apoptosis, accompanied by activation of tyrosine and stress kinases, phosphorylation of FAK and paxillin, and elevation of intracellular free [Ca2+]. The apoptosis is attenuated by inhibition of transcription or translation, by inhibition of tyrosine kinases, or by intracellular Ca2+ buffering. These data suggest that clinical use of the osmotic diuretic mannitol may exert direct deleterious effects on vascular endothelium.

Animals↗

PubMed1998

Immunolocalization and tissue-specific splicing of AE2 anion exchanger in mouse kidney.

In this study, an epitope-unmasking technique was used to immunolocalize AE2 anion exchanger polypeptide to basolateral plasma membranes of tubular epithelial cells in mouse kidney. Kidney AE2 immunostaining in mouse kidney was less prominent than in rat, consistent with the relative levels of AE2 mRNA and polypeptide in these two species. Glomeruli showed faint but consistent AE2 immunostaining, whereas proximal tubules were generally unstained. Macula densa epithelial cells displayed bright AE2 immunostaining, and cortical thick limbs were stained at a lower intensity. AE2 immunostaining was weak or absent in type B intercalated cells and principal cells of the cortical collecting duct, but increased in intensity in principal cells of the inner stripe of the outer medulla. AE2 staining in medullary thick limbs was also of greater intensity than in cortical thick limbs. AE2 staining was strong and uniform in the epithelial cells of the inner medullary collecting duct, and in epithelial cells of the papillary surface, the ureter, and the urinary bladder. Extratubular and epithelial cells of the inner medulla also showed punctate intracellular AE2 staining in a Golgi-like distribution that, in contrast to cell surface staining, was sodium dodecyl sulfate-sensitive. Golgi localization of AE2 epitope was confirmed by immunoperoxidase electron microscopy. Reverse transcription-PCR analysis of mouse kidney RNA detected AE2a, AE2b, and an AE2c2 transcript, but an AE2c1 transcript was absent. Unlike in rat, the mouse AE2c2 mRNA splice variant encoded a polypeptide with a novel predicted N-terminal amino acid sequence.

Amino Acid Sequence↗

PubMed1997

The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances. Demonstration of efficacy in intact rabbit colon and in an in vivo mouse model of cholera.

The antifungal antibiotic clotrimazole (CLT) blocks directly and with high potency the Ca2+-activated K+ channels of human erythrocytes, erythroleukemia cells, and ferret vascular smooth muscle cells. We recently reported that CLT inhibits Cl- secretion in human intestinal T84 cells, likely by affecting K+ transport (Rufo, P.A., L. Jiang, S.J. Moe, C. Brugnara, S.L. Alper, and W.I. Lencer. 1996. J. Clin. Invest. 98:2066-2075). To determine if CLT had direct effects on K+ conductances in T84 cells, we selectively permeabilized apical membranes of confluent T84 cell monolayers using the ionophore amphotericin B. This technique permits direct measurement of basolateral K+ transport. We found that CLT and a stable des-imidazolyl derivative inhibited directly two pharmacologically distinct basolateral membrane K+conductances, but had no effect on apical membrane Cl- conductances. The effects of CLT on Cl- secretion were also examined in intact tissue. CLT inhibited forskolin-induced Cl- secretion in rabbit colonic mucosal sheets mounted in Ussing chambers by 91%. CLT also inhibited cholera toxin-induced intestinal Cl- secretion in intact mice by 94%. These data provide direct evidence that CLT blocks Cl- secretion in intestinal T84 cells by inhibition of basolateral K+ conductances, and show that CLT inhibits salt and water secretion from intact tissue in vitro and in vivo. The results further support the suggestion that CLT and its metabolites may show clinical efficacy in the treatment of secretory diarrheas of diverse etiologies.

Animals↗

PubMed1997

Functional consequences of mutations in the transmembrane domain and the carboxy-terminus of the murine AE1 anion exchanger.

We have characterized mouse AE1-mediated 36Cl- influx and surface AE1 polypeptide expression in Xenopus oocytes injected with cRNA encoding two classes of loss-of-function mutants. The first arose spontaneously. Chimeric mutants constructed with a functional AE1 cDNA localized the site of spontaneous mutation to the transmembrane domain, and DNA sequencing revealed two missense mutations encoding the double-mutant polypeptide V728F/M7301. Each mutation individually produced only partial loss of AE1 transport activity, and coexpression of the individual mutants did not restore full activity. The functional changes produced by the mutations correlated with reduced fractional accumulation of polypeptides at the oocyte surface. The V728F/M7301 polypeptide expressed in mammalian cells displayed complete endoH resistance and rapid degradation. We also examined the effect on AE1 function of engineered removal of its hydrophilic carboxy-terminus. Both delta(c)890 and the internal deletion delta(c)890-917 were functionally inactive in Xenopus oocytes. Lack of transport activity correlated with lack of detectable polypeptide accumulation at the oocyte surface. Coexpression with wt AE1 of some, but not all, of these AE1 mutants partially suppressed wt AE1-mediated 36Cl- uptake. In contrast, coexpression with wt AE1 of soluble N-terminal AE1 fragments was not inhibitory.

Animals↗

PubMed1997

Routes of Cl- transport across the trophectoderm of the mouse blastocyst.

The blastocyst stage of embryo development is characterized by a fluid-filled cavity called the blastocoel. Blastocoel formation requires vectorial Na+ and Cl- transport and the accompanying osmotic accumulation of fluid. We found under conditions of low external Cl- that inhibitors of Cl- transport mechanisms inhibited blastocoel expansion, indicating a possible transcellular route for Cl- uptake across the outer epithelial layer (the trophectoderm). Using the Cl--sensitive fluorophore, N-(6-methoxyquinolyl)acetoethyl ester, we found that Cl- efflux from the blastocoel can occur via pathways with properties that resemble both HCO-3/Cl- exchange and Cl- channels, as well as by another yet uncharacterized pathway. In contrast, Cl- re-uptake into Cl--depleted blastocoels (the physiologically relevant direction for Cl- transport during blastocoel expansion) occurred only via the channel-like mechanism. Patch-clamp recordings detected a component of current carried by apical Cl- channels. Intracellular pH measurements during external Cl- removal detected HCO-3/Cl- exchange activity in collapsed blastocysts but little in intact blastocysts, suggesting predominantly basolateral HCO-3/Cl- exchange activity. This was corroborated by the immunolocalization of the AE2 isoform of HCO-3/Cl- exchanger to the basolateral surface of the trophectoderm. Thus, it appears that Cl- transport into the blastocoel may occur via apical Cl- channels, while efflux also involves a basolateral HCO-3/Cl- exchanger.

Animals↗

PubMed1997

The antifungal imidazole clotrimazole and its major in vivo metabolite are potent blockers of the calcium-activated potassium channel in murine erythroleukemia cells.

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca2+)-activated 86Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of 125I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell KCa2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca2+ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca2+. The extent of inhibition of whole cell MEL KCa2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single KCa2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca2+ currents in PC12 cells >> Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single KCa2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells.

Animals↗