Lianwei Jiang

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 inXenopus 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.

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 ∼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.

Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes

Mutations in the human SLC26A3 gene, also known as down‐regulated in adenoma (hDRA), cause autosomal recessive congenital chloride‐losing diarrhoea (CLD). hDRA expressed in Xenopus oocytes mediated bidirectional Cl−‐Cl− and Cl−‐HCO3− exchange. In contrast, transport of oxalate was low, and transport of sulfate and of butyrate was undetectable. Two CLD missense disease mutants of hDRA were nonfunctional in oocytes. Truncation of up to 44 C‐terminal amino acids from the putatively cytoplasmic C‐terminal hydrophilic domain left transport function unimpaired, but deletion of the adjacent STAS (sulfate transporter anti‐sigma factor antagonist) domain abolished function. hDRA‐mediated Cl− transport was insensitive to changing extracellular pH, but was inhibited by intracellular acidification and activated by NH4+ at acidifying concentrations. These regulatory responses did not require the presence of either hDRAs N‐terminal cytoplasmic tail or its 44 C‐terminal amino acids, but they did require more proximate residues of the C‐terminal cytoplasmic domain. Although only weakly sensitive to inhibition by stilbenes, hDRA was inhibited with two orders of magnitude greater potency by the anti‐inflammatory drugs niflumate and tenidap. cAMP‐insensitive Cl−‐HCO3− exchange mediated by hDRA gained modest cAMP sensitivity when co‐expressed with cystic fibrosis transmembrane conductance regulator (CFTR). Despite the absence of hDRA transcripts in human cell lines derived from CFTR patients, DRA mRNA was present at wild‐type levels in proximal colon and nearly so in the distal ileum of CFTR(‐/‐) mice. Thus, pharmacological modulation of DRA might be a useful adjunct treatment of cystic fibrosis.

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.

The Cytoplasmic and Transmembrane Domains of AE2 Both Contribute to Regulation of Anion Exchange by pH

We have compared regulation by pH of AE1 (band 3)- and AE2-mediated Cl uptake into Xenopus oocytes. Cl influx was assayed at varying extracellular pH (pH) values between 9.0 and 5.0 under conditions in which corresponding intracellular pH (pH) values were at or near steady-state. Wild type (WT) AE1 displayed a broad convex pH versus activity curve, with peak activity at pH 7.0 and 63% of maximal activity at pH 5.0. In contrast, WT AE2 displayed a steep pH versus activity curve, with peak activity at pH9.0 and full suppression at pH 5.0. The structural basis of these differing pH sensitivities was examined by expression of cRNAs encoding chimeric and truncated proteins. Mutant polypeptides were expressed in oocytes and detected at the cell surface. The AE2/AE1 polypeptide displayed a broad pH versus activity curve similar to that of WT AE1. In contrast, the AE1/AE2 polypeptide displayed a steep pH versus activity curve, which was shifted toward acid pH values from that of WT AE2 by 0.69 ± 0.04 pH units. Moreover, whereas the pH versus activity curves of AE2 Δ99 and WT AE2 were indistinguishable, AE2 Δ510 exhibited a pH versus activity curve acid-shifted from that of WT AE2 by 0.66 ± 0.13 pH units (indistinguishable from that of AE1/AE2). The data suggest that a pH sensor resides within the transmembrane region of AE2. The affinity for protons of this pH sensor is influenced by a modifier site located between residues 99 and 510 of the N-terminal cytoplasmic domain of AE2. Acidification of oocytes with acetate suggested that pH accounted for some but not all of the measured pH dependence of AE2.

The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells.

Clotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals.

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.

c-Myb–Dependent Cell Cycle Progression and Ca2+ Storage in Cultured Vascular Smooth Muscle Cells

Considerable controversy surrounds the role of the c-myb proto-oncogene in vascular smooth muscle cells (VSMCs). Previous investigations using antisense approaches have suggested a relationship between c-myb expression, cell cycle progression, and cytoplasmic Ca2+ concentration ([Ca2+]cyt). However, the ability of certain antisense oligonucleotides to bind and inactivate growth factors allows alternative explanations. To define more specifically the role of c-Myb in cultured VSMCs (SVE and A10 cell lines), we have generated stable cell clones expressing a dominant-negative c-Myb lacking critical elements of the DNA binding domain (delta5-SVE) and transiently transfected cell populations (GRE-MEn-SVE and GRE-MEn-A10) expressing a glucocorticoid-inducible chimeric protein that targets the Drosophila Engrailed repressor domain to c-Myb-responsive promoters. The delta5-SVE clones and GRE-MEn cell populations exhibit a 60% reduction in mean intracellular c-Myb activity, as measured by cotransfection assays with a c-Myb-responsive reporter, a 42% decrease in the mean S phase entry of growth-arrested (G[0]) cells after serum stimulation, and a 36% inhibition of mean cell proliferation over 4 days. These cells also display 28% (34-nmol/L) and 30% (42-nmol/L) reductions in mean [Ca2+]cyt at G(0) and at the G1/S interface, respectively, as well as significant reductions in the peak [Ca2+]cyt responses to thapsigargin (5 micromol/L) and caffeine (10 mmol/L). These latter reductions in operationally defined Ca2+ pools were observed both at different stages of the cell cycle and after transient induction of the dominant-interfering construct, suggesting that c-Myb regulates these releasable Ca2+ stores independent of its effects on cell cycle progression.

Deficient Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer

Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange activity mediated by the AE1 anion exchanger is reduced by carbonic anhydrase II (CA2) inhibition or by prevention of CA2 binding to the AE1 C-terminal cytoplasmic tail. This type of AE1 inhibition is thought to represent reduced metabolic channeling of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} to the intracellular \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} binding site of AE1. To test the hypothesis that CA2 binding might itself allosterically activate AE1 in Xenopus oocytes, we compared Cl-/Cl- and Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange activities of AE1 polypeptides with truncation and missense mutations in the C-terminal tail. The distal renal tubular acidosis-associated AE1 901X mutant exhibited both Cl-/Cl- and Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange activities. In contrast, AE1 896X, 891X, and AE1 missense mutants in the CA2 binding site were inactive as Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchangers despite exhibiting normal Cl-/Cl- exchange activities. Co-expression of CA2 enhanced wild-type AE1-mediated Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange, but not Cl-/Cl- exchange. CA2 co-expression could not rescue Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange activity in AE1 mutants selectively impaired in Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange. However, co-expression of transport-incompetent AE1 mutants with intact CA2 binding sites completely rescued Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange by the AE1 truncation mutant 896X, despite preservation of the latters core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer.

pHi and serum regulate AE2-mediated Cl-/HCO3-, exchange in CHOP cells of defined transient transfection status

Anion exchanger (AE) protein-mediated anion exchange contributes to regulation of intracellular pH (pHi), Cl- concentration, and volume in vertebrate cells. We have extended the functional characterization of recombinant AE2-mediated Cl-/HCO3- exchange in single Chinese hamster ovary cells stably transfected with the polyoma large T antigen (CHOP cells) of defined transient transfection status using a novel surface marker coexpression vector. Marker expression and detection had minimal effect on the low endogenous Cl-/HCO3- exchange activity of CHOP cells, whereas coexpression of marker with AE2 elevated CHOP cell Cl-/HCO3- exchange activity 16-fold. Between pHi of 7.3 and 7.8, AE2-mediated flux of proton equivalents was activated > 11-fold by increasingly alkaline pHi without reaching saturation. This activation may be secondary to allosteric effects of pHi on AE2, in parallel with the obligatory increase in substrate intracellular HCO3- concentration. Nominal removal of CO2/HCO3- reduced AE2 activity by 90%. Addition of 10% calf serum slowly activated AE2 activity severalfold. This activation was slowly reversed after serum removal. Surface marker coexpression vectors improve both the efficiency and reliability of studies of recombinant protein function for a wide range of single cell assays in many cell types.