ABSTRACT
Mutations in the genes encoding the mechanosensitive cation channels PIEZO1 and PIEZO2 are responsible for multiple hereditary human diseases. Loss-of-function mutations in the human PIEZO1 gene cause autosomal recessive congenital lymphatic dysplasia. Gain-of-function mutations in the human PIEZO1 gene cause the autosomal dominant hemolytic anemia, hereditary xerocytosis (also known as dehydrated stomatocytosis). Loss-of-function mutations in the human PIEZO2 gene cause an autosomal recessive syndrome of muscular atrophy with perinatal respiratory distress, arthrogryposis, and scoliosis. Gain-of-function mutations in the human PIEZO2 gene cause three clinical types of autosomal dominant distal arthrogryposis. This chapter will review the hereditary diseases caused by mutations in the PIEZO genes and will discuss additional physiological systems in which PIEZO channel dysfunction may contribute to human disease pathophysiology.
Subject(s)
Disease/genetics , Ion Channels/genetics , Ion Channels/metabolism , Animals , Humans , Loss of Function MutationABSTRACT
The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 â¼ BCLW > BCLXL â¼ BCL2A1 â« BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na(+) by K(+) also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca(2+) and Cl(-) fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins.
Subject(s)
Apolipoproteins/biosynthesis , Apolipoproteins/toxicity , Lipoproteins, HDL/biosynthesis , Lipoproteins, HDL/toxicity , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Amino Acid Sequence , Animals , Apolipoprotein L1 , Apolipoproteins/genetics , Female , Humans , Lipoproteins, HDL/genetics , Mice , Molecular Sequence Data , Protein Structure, Tertiary/physiology , Proto-Oncogene Proteins c-bcl-2/genetics , Xenopus laevisABSTRACT
The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABA(A) receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl(-) influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K(+)/Cl(-) cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K(+)/Cl(-) cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine-threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl(-) levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.
Subject(s)
Neurons/metabolism , Suprachiasmatic Nucleus/metabolism , Symporters/metabolism , Animals , Gastrin-Releasing Peptide/metabolism , Male , Neurons/ultrastructure , Protein Kinases/metabolism , Rats , Rats, Sprague-Dawley , Sodium-Potassium-Chloride Symporters/metabolism , Solute Carrier Family 12, Member 2 , Suprachiasmatic Nucleus/ultrastructure , Vasoactive Intestinal Peptide/metabolism , Vasopressins/metabolism , K Cl- CotransportersABSTRACT
Pancreatic ductal epithelium produces a HCO(3)(-)-rich fluid. HCO(3)(-) transport across ductal apical membranes has been proposed to be mediated by both SLC26-mediated Cl(-)/HCO(3)(-) exchange and CFTR-mediated HCO(3)(-) conductance, with proportional contributions determined in part by axial changes in gene expression and luminal anion composition. In this study we investigated the characteristics of apical Cl(-)/HCO(3)(-) exchange and its functional interaction with Cftr activity in isolated interlobular ducts of guinea pig pancreas. BCECF-loaded epithelial cells of luminally microperfused ducts were alkalinized by acetate prepulse or by luminal Cl(-) removal in the presence of HCO(3)(-)-CO(2). Intracellular pH recovery upon luminal Cl(-) restoration (nominal Cl(-)/HCO(3)(-) exchange) in cAMP-stimulated ducts was largely inhibited by luminal dihydro-DIDS (H(2)DIDS), accelerated by luminal CFTR inhibitor inh-172 (CFTRinh-172), and was insensitive to elevated bath K(+) concentration. Luminal introduction of CFTRinh-172 into sealed duct lumens containing BCECF-dextran in HCO(3)(-)-free, Cl(-)-rich solution enhanced cAMP-stimulated HCO(3)(-) secretion, as calculated from changes in luminal pH and volume. Luminal Cl(-) removal produced, after a transient small depolarization, sustained cell hyperpolarization of approximately 15 mV consistent with electrogenic Cl(-)/HCO(3)(-) exchange. The hyperpolarization was inhibited by H(2)DIDS and potentiated by CFTRinh-172. Interlobular ducts expressed mRNAs encoding CFTR, Slc26a6, and Slc26a3, as detected by RT-PCR. Thus Cl(-)-dependent apical HCO(3)(-) secretion in pancreatic duct is mediated predominantly by an Slc26a6-like Cl(-)/HCO(3)(-) exchanger and is accelerated by inhibition of CFTR. This study demonstrates functional coupling between Cftr and Slc26a6-like Cl(-)/HCO(3)(-) exchange activity in apical membrane of guinea pig pancreatic interlobular duct.
Subject(s)
Bicarbonates/metabolism , Chloride-Bicarbonate Antiporters/physiology , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Pancreatic Ducts/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/analogs & derivatives , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Benzoates/pharmacology , Chloride-Bicarbonate Antiporters/antagonists & inhibitors , Chlorides/metabolism , Colforsin/pharmacology , Colon/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Cytosol/drug effects , Cytosol/metabolism , Gene Expression/genetics , Guinea Pigs , Hydrogen-Ion Concentration/drug effects , Kidney/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Pancreatic Ducts/drug effects , Perfusion , Thiazolidines/pharmacologyABSTRACT
The widely expressed anion exchanger polypeptide AE2/SLC4A2 is acutely inhibited by acidic intracellular (pH(i)), by acidic extracellular pH (pH(o)), and by the calmodulin inhibitor, calmidazolium, whereas it is acutely activated by NH(4)(+). The homologous erythroid/kidney AE1/SLC4A1 polypeptide is insensitive to these regulators. Each of these AE2 regulatory responses requires the presence of AE2's C-terminal transmembrane domain (TMD). We have now measured (36)Cl(-) efflux from Xenopus oocytes expressing bi- or tripartite AE2-AE1 chimeras to define TMD subregions in which AE2-specific sequences contribute to acute regulation. The chimeric AE polypeptides were all functional at pH(o) 7.4, with the sole exception of AE2((1-920))/AE1((613-811))/AE2((1120-1237)). Reciprocal exchanges of the large third extracellular loops were without effect. AE2 regulation by pH(i), pH(o) and NH(4)(+) was retained after substitution of C-terminal AE2 amino acids 1120-1237 (including the putative second re-entrant loop, two TM spans and the cytoplasmic tail) with the corresponding AE1 sequence. In contrast, the presence of this AE2 C-terminal sequence was both necessary and sufficient for inhibition by calmidazolium. All other tested TMD substitutions abolished AE2 pH(i) sensitivity, abolished or severely attenuated sensitivity to pH(o) and removed sensitivity to NH(4)(+). Loss of AE2 pH(i) sensitivity was not rescued by co-expression of a complementary AE2 sequence within separate full-length chimeras or AE2 subdomains. Thus, normal regulation of AE2 by pH and other ligands requires AE2-specific sequence from most regions of the AE2 TMD, with the exceptions of the third extracellular loop and a short C-terminal sequence. We conclude that the individual TMD amino acid residues previously identified as influencing acute regulation of AE2 exert that influence within a regulatory structure requiring essential contributions from multiple regions of the AE2 TMD.
Subject(s)
Anion Transport Proteins/metabolism , Antiporters/metabolism , Imidazoles/pharmacology , Quaternary Ammonium Compounds/metabolism , Amino Acid Sequence , Animals , Anion Exchange Protein 1, Erythrocyte/metabolism , Anion Transport Proteins/antagonists & inhibitors , Anion Transport Proteins/chemistry , Antiporters/antagonists & inhibitors , Antiporters/chemistry , Cell Membrane/metabolism , Chloride-Bicarbonate Antiporters , Codon, Nonsense , Female , Hydrogen-Ion Concentration , Mice , Oocytes/metabolism , Peptides/metabolism , Protein Isoforms/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , SLC4A Proteins , Xenopus laevisABSTRACT
The ubiquitous AE2/SLC4A2 anion exchanger is acutely and independently regulated by intracellular (pH(i)) and extracellular pH (pH(o)), whereas the closely related AE1/SLC4A1 of the red cell and renal intercalated cell is relatively pH-insensitive. We have investigated the contribution of nonconserved charged residues within the C-terminal transmembrane domain (TMD) of AE2 to regulation by pH through mutation to the corresponding AE1 residues. AE2-mediated Cl(-)/Cl(-) exchange was measured as 4,4'-di-isothiocyanatostilbene-2,2'-disulfonic acid-sensitive (36)Cl(-) efflux from Xenopus oocytes by varying pH(i) at constant pH(o), and by varying pH(o) at near-constant pH(i). All mutations of nonconserved charged residues of the AE2 TMD yielded functional protein, but mutations of some conserved charged residues (R789E, R1056A, R1134C) reduced or abolished function. Individual mutation of AE2 TMD residues R921, F922, P1077, and R1107 exhibited reduced pH(i) sensitivity compared to wt AE2, whereas TMD mutants K1153R, R1155K, R1202L displayed enhanced sensitivity to acidic pH(i). In addition, pH(o) sensitivity was significantly acid- shifted when nonconserved AE2 TMD residues E981, K982, and D1075 were individually converted to the corresponding AE1 residues. These results demonstrate that multiple conserved charged residues are important for basal transport function of AE2 and that certain nonconserved charged residues of the AE2 TMD are essential for wild-type regulation of anion exchange by pH(i) and pH(o).
Subject(s)
Anion Transport Proteins/chemistry , Anion Transport Proteins/metabolism , Antiporters/chemistry , Antiporters/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Anion Exchange Protein 1, Erythrocyte/chemistry , Anion Exchange Protein 1, Erythrocyte/genetics , Anion Exchange Protein 1, Erythrocyte/metabolism , Anion Transport Proteins/genetics , Antiporters/genetics , Chloride-Bicarbonate Antiporters , Chlorides/metabolism , Conserved Sequence , Female , Hydrogen-Ion Concentration , In Vitro Techniques , Ion Exchange , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes/metabolism , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SLC4A Proteins , Sequence Homology, Amino Acid , XenopusABSTRACT
Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pH(i)) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl(-)/Cl(-) and Cl(-)/HCO(3)(-) exchange are inhibited by acid pH(i) or extracellular pH (pH(o)). We have investigated the importance to pH sensitivity of the eight histidine (His) residues within the AE2 COOH-terminal transmembrane domain (TMD). Wild-type mouse AE2-mediated Cl(-)/Cl(-) exchange, measured as DIDS-sensitive (36)Cl(-) efflux from Xenopus oocytes, was experimentally altered by varying pH(i) at constant pH(o) or varying pH(o). Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal (36)Cl(-) efflux at pH(o) 7.4 and acid shifted the pH(o) vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl(-) transport activity with retention of pH(o) sensitivity. In contrast to the effects of DEPC on wild-type AE2, pH(o) sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pH(i), pH(i) sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pH(i) and pH(o).
Subject(s)
Anion Transport Proteins/physiology , Antiporters/physiology , Bicarbonates/metabolism , Chlorides/metabolism , Histidine/metabolism , Animals , Anion Transport Proteins/genetics , Antiporters/genetics , Chloride-Bicarbonate Antiporters , Female , Histidine/genetics , Hydrogen-Ion Concentration , In Vitro Techniques , Ion Transport , Mutation , Oocytes/metabolism , Protein Structure, Tertiary , SLC4A Proteins , XenopusABSTRACT
Chronic intermittent hypoxia (CIH) is a component of several disease states, including obstructive sleep apnea, which results in neurocognitive and cardiovascular morbidity. Because chronic hypoxia can induce changes in metabolism and pH homeostasis, we hypothesized that CIH induces changes in the expression of acid-base transporters. Two- to three-day-old mice, exposed to alternating cycles of 2 min of hypoxia (6.0-7.5% O2) and 3 min of normoxia (21% O2) for 8 h/day for 28 days, demonstrated decreases in specific acid-base transport protein expression in most of the central nervous system (CNS). Sodium/hydrogen exchanger isoform 1 (NHE1) and sodium-bicarbonate cotransporter expression were decreased in all regions of the CNS but especially so in the cerebellum. NHE3, which is only expressed in the cerebellum, was also significantly decreased. Anion exchanger 3 protein was decreased in most brain regions, with the decrease being substantial in the hippocampus. These results indicate that CIH induces downregulation of the major acid-extruding transport proteins, NHE1 and sodium-bicarbonate cotransporter, in particular regions of the CNS. This downregulation in acid-extruding capacity may render neurons more prone to acidity and possibly to injury during CIH, especially in the cerebellum and hippocampus. Alternatively, it is possible that O2 consumption in these regions is decreased after CIH, with consequential downregulation in the expression of certain cellular proteins that may be less needed under such circumstances.
Subject(s)
Bicarbonates/metabolism , Carrier Proteins/metabolism , Central Nervous System/metabolism , Hypoxia/metabolism , Sodium-Hydrogen Exchangers/biosynthesis , Acid-Base Equilibrium/physiology , Animals , Blotting, Western , Cerebellum/metabolism , Chronic Disease , Female , Hippocampus/metabolism , Hypoxia/enzymology , Mice , Pregnancy , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/metabolismABSTRACT
We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).
Subject(s)
Anion Transport Proteins , Antiporters , Chloride-Bicarbonate Antiporters/metabolism , Conserved Sequence/physiology , Cytoplasm/metabolism , Ion Transport/physiology , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Acids/pharmacokinetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Biological Transport, Active/physiology , Chloride-Bicarbonate Antiporters/chemistry , Female , Hydrogen-Ion Concentration , Kinetics , Mutagenesis, Site-Directed , Mutation, Missense , Protein Structure, Tertiary/physiology , SLC4A Proteins , Structure-Activity Relationship , XenopusABSTRACT
In the course of studying the hypertonicity-activated ion transporters in Xenopus oocytes, we found that activation of endogenous oocyte Na(+)/H(+) exchange activity (xoNHE) by hypertonic shrinkage required Cl(-), with an EC(50) for bath [Cl(-)] of approximately 3mM. This requirement for chloride was not supported by several nonhalide anions and was not shared by xoNHE activated by acid loading. Hypertonicity-activated xoNHE exhibited an unusual rank order of inhibitory potency among amiloride derivatives and was blocked by Cl(-) transport inhibitors. Chelation of intracellular Ca(2+) by injection of EGTA blocked hypertonic activation of xoNHE, although many inhibitors of Ca(2+)-related signaling pathways were without inhibitory effect. Hypertonicity activated oocyte extracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors of neither ERK1/2 nor p38 prevented hypertonic activation of xoNHE. However, hypertonicity also stimulated a Cl(-)-dependent increase in c-Jun NH(2)-terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE but not activation by acid loading. We conclude that hypertonic activation of Na(+)/H(+) exchange in Xenopus oocytes requires Cl(-) and is mediated by activation of JNK.
Subject(s)
Chlorides/metabolism , Mitogen-Activated Protein Kinases/metabolism , Sodium-Hydrogen Exchangers/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Anions/pharmacology , Anthracenes/pharmacology , Calcium/metabolism , Chelating Agents/pharmacology , Diuretics/pharmacology , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Female , Hydrogen/metabolism , Hydrogen-Ion Concentration , Hypertonic Solutions/chemistry , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Oocytes/physiology , Osmolar Concentration , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sodium/metabolism , Sodium Radioisotopes/metabolism , Xenopus laevisABSTRACT
The role of intracellular pH (pH(i)) in regulation of AE2 function in Xenopus oocytes remains unclear. We therefore compared AE2-mediated (36)Cl(-) efflux from Xenopus oocytes during imposed variation of extracellular pH (pH(o)) or variation of pH(i) at constant pH(o). Wild-type AE2-mediated (36)Cl(-) efflux displayed a steep pH(o) vs. activity curve, with pH(o(50)) = 6.91 +/- 0.04. Sequential NH(2)-terminal deletion of amino acid residues in two regions, between amino acids 328 and 347 or between amino acids 391 and 510, shifted pH(o(50)) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocyte pH(i) at constant pH(o) and were shown to be neither substrates nor inhibitors of AE2-mediated Cl(-) transport. At constant pH(o), AE2 was inhibited by intracellular acidification and activated by intracellular alkalinization. Our data define structure-function relationships within the AE2 NH(2)-terminal cytoplasmic domain, which demonstrates distinct structural requirements for AE2 regulation by intracellular and extracellular protons.
Subject(s)
Anion Transport Proteins , Antiporters , Hydrogen-Ion Concentration , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Acids/pharmacology , Animals , Bicarbonates/pharmacokinetics , Butyrates/pharmacokinetics , Carbon Radioisotopes , Chlorides/pharmacokinetics , Cytoplasm/metabolism , Membrane Proteins/genetics , Microelectrodes , Mutagenesis/physiology , Oocytes/physiology , Protein Structure, Tertiary , Protons , Radioisotopes , SLC4A Proteins , XenopusABSTRACT
Mg is an important determinant of erythrocyte cation transport system(s) activity. We investigated cation transport in erythrocytes from mice bred for high (MGH) and low (MGL) Mg levels in erythrocytes and plasma. We found that K-Cl cotransport activity was higher in MGL than in MGH erythrocytes, and this could explain their higher mean corpuscular hemoglobin concentration, median density, and reduced cell K content. Although mouse KCC1 protein abundance was comparable in MGL and MGH erythrocytes, activities of Src family tyrosine kinases were higher in MGH than in MGL erythrocytes. In contrast, protein phosphatase (PP) isoform 1 alpha (PP1 alpha) enzymatic activity, which has been suggested to play a positive regulatory role in K-Cl cotransport, was lower in MGH than in MGL erythrocytes. Additionally, we found that the Src family kinase c-Fgr tyrosine phosphorylates PP1 alpha in vitro. These findings suggest that in vivo downregulation of K-Cl cotransport activity by Mg is mediated by enhanced Src family kinase activity, leading to inhibition of the K-Cl cotransport stimulator PP1.
Subject(s)
Carrier Proteins/metabolism , Erythrocytes/metabolism , Magnesium/metabolism , Symporters , Animals , Biological Transport/drug effects , Biological Transport/physiology , Cations/metabolism , Enzyme Inhibitors/pharmacology , Ethylmaleimide/pharmacology , Mice , Mice, Inbred AKR , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Okadaic Acid/pharmacology , Oxidation-Reduction , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Proto-Oncogene Proteins/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Species Specificity , Staurosporine/pharmacology , src-Family Kinases/metabolism , K Cl- CotransportersABSTRACT
K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.
Subject(s)
Chlorides/metabolism , Potassium/metabolism , Symporters/chemistry , Animals , Cytoplasm/chemistry , Humans , Mice , Mutation , Rats , Structure-Activity Relationship , Symporters/genetics , Symporters/physiology , K Cl- CotransportersABSTRACT
Transgenic plants overexpressing the vacuolar H(+)-pyrophosphatase are much more resistant to high concentrations of NaCl and to water deprivation than the isogenic wild-type strains. These transgenic plants accumulate more Na(+) and K(+) in their leaf tissue than the wild type. Moreover, direct measurements on isolated vacuolar membrane vesicles derived from the AVP1 transgenic plants and from wild type demonstrate that the vesicles from the transgenic plants have enhanced cation uptake. The phenotypes of the AVP1 transgenic plants suggest that increasing the vacuolar proton gradient results in increased solute accumulation and water retention. Presumably, sequestration of cations in the vacuole reduces their toxic effects. Genetically engineered drought- and salt-tolerant plants could provide an avenue to the reclamation of farmlands lost to agriculture because of salinity and a lack of rainfall.
Subject(s)
Arabidopsis/genetics , Proton Pumps/genetics , Pyrophosphatases/genetics , Abscisic Acid/pharmacology , Arabidopsis/drug effects , Arabidopsis/enzymology , Calcimycin/pharmacology , Calcium/metabolism , Calcium/pharmacology , Kinetics , Phenotype , Plants, Genetically Modified , Plasmids , Potassium/metabolism , Restriction Mapping , Sodium/metabolism , Solutions , Vacuoles/metabolism , Water/metabolismABSTRACT
1. The anion exchanger isoform 2 (AE2) gene encodes three subtypes (AE2a, b and c), which have different N-termini and tissue distributions. AE2 is expressed at high levels in the stomach, where it is thought to mediate basolateral base exit during acid production. The present study investigated if the three AE2 subtypes are differentially expressed and regulated in different cell types within the gastric mucosa. 2. The cloning strategy to obtain rabbit AE2a, b and c cDNAs combined genomic PCR and RT-PCR based on primers deduced from the rat sequences. Semiquantitative RT-PCR using homologous primers revealed much higher AE2 mRNA expression in rabbit parietal cells (PCs) than in mucous cells (MCs). The subtype expression pattern was AE2b >> AE2c > or = AE2a in PCs and AE2a >AE2b >> AE2c in MCs. Sequence analysis revealed the presence of a highly conserved protein kinase C (PKC) consensus sequence in the AE2a alternative N-terminus. 3. Maximal Cl(-)-HCO(3)(-) exchange rates, measured fluorometrically in BCECF-loaded cultured gastric cells, were much higher in PCs than MCs. PKC activation by phorbol ester stimulated maximal Cl(-)-HCO(3)(-) exchange rates in MCs but not in PCs, whereas forskolin had no effect in each cell type. 4. In summary, rabbit PCs and MCs, which originate from the same gastric stem cell population, display a completely different AE2 subtype expression pattern. Therefore, AE2 subtype expression is not organ specific but cell type specific. The different regulation of anion exchange in parietal and mucous cells suggests that AE2 subtypes may be differentially regulated.
Subject(s)
Anion Transport Proteins , Antiporters , Gastric Mucosa/metabolism , Membrane Proteins/metabolism , Parietal Cells, Gastric/metabolism , Amino Acid Sequence/genetics , Animals , Blotting, Northern , Gastric Mucosa/cytology , Immunoblotting , Immunohistochemistry , Membrane Proteins/genetics , Microscopy, Fluorescence , Molecular Sequence Data , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , SLC4A Proteins , Tissue DistributionABSTRACT
Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 microg/ml) and angiostatin (5 microg/ml) elicited transient, approximately threefold increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca(2+)](i) responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca(2+) signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP(3)) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca(2+)](i), and endostatin rapidly elevated endothelial cell IP(3) levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca(2+) signal. Removal of extracellular Ca(2+) inhibited the endostatin-induced rise in [Ca(2+)](i), as did a subset of Ca(2+)-entry inhibitors. Peak Ca(2+) responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca(2+)](i) in response to vascular endothelial growth factor or to fibroblast growth factor by approximately 70%. Intracellular Ca(2+) signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Calcium Signaling/physiology , Collagen/pharmacology , Endothelium, Vascular/physiology , Peptide Fragments/pharmacology , Plasminogen/pharmacology , Angiostatins , Animals , Aorta, Thoracic , COS Cells , Calcium/metabolism , Calcium Channels , Calcium Signaling/drug effects , Cattle , Cell Line , Cells, Cultured , Chlorocebus aethiops , Endostatins , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Humans , Imidazoles/pharmacology , Inositol 1,4,5-Trisphosphate Receptors , Macrocyclic Compounds , Mice , Oxazoles/pharmacology , Pertussis Toxin , Pulmonary Artery , Pyrimidines/pharmacology , Pyrrolidinones/pharmacology , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Recombinant Proteins/pharmacology , Thapsigargin/pharmacology , Transfection , Type C Phospholipases/metabolism , Virulence Factors, Bordetella/pharmacologyABSTRACT
Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are thought to play a critical role in the pathogenesis of both sporadic and VHL disease-associated clear-cell renal carcinomas (RCC). Differential display-PCR identified the AE2 anion exchanger as a candidate VHL target gene. AE2 mRNA and polypeptide levels were approximately threefold higher in 786-O VHL cells than in 786-O Neo cells. In contrast, Cl(-)/HCO(3)(-) exchange activity in 786-O VHL cells was 50% lower than in 786-O Neo cells. Since resting intracellular pH (pH(i)) values were indistinguishable, we postulated that Na(+)/H(+) exchange activity (NHE) might be similarly reduced in 786-O VHL cells. NHE-mediated pH(i) recovery from acid load was less than 50% that in 786-O Neo cells, whereas hypertonicity-stimulated, amiloride-sensitive NHE was indistinguishable in the two cell lines. The NHE3 mRNA level was higher in 786-O VHL than 786-O Neo cells, but NHE1 mRNA levels did not differ. AE2 and NHE3 are the first transcripts reported to be upregulated by pVHL. Elucidation of mechanisms responsible for downregulation of both ion exchange activities will require further investigation.
Subject(s)
Anion Transport Proteins , Carcinoma, Renal Cell/metabolism , Genes, Tumor Suppressor , Ligases , Membrane Proteins/metabolism , Proteins/physiology , Sodium-Hydrogen Exchangers/metabolism , Tumor Suppressor Proteins , Ubiquitin-Protein Ligases , Ammonium Chloride/pharmacology , Antiporters/genetics , Antiporters/metabolism , Carbonic Anhydrases/genetics , Carcinoma, Renal Cell/genetics , Chloride-Bicarbonate Antiporters , Down-Regulation , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Hydrogen-Ion Concentration , Hypertonic Solutions , Membrane Proteins/genetics , Proteins/genetics , RNA, Neoplasm/biosynthesis , SLC4A Proteins , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/genetics , Tumor Cells, Cultured , Up-Regulation , Von Hippel-Lindau Tumor Suppressor ProteinABSTRACT
We have studied the expression and localization of several H(+) and HCO(3)(-) transporters, whose presence in the rat pancreas is still unclear. The Cl(-)/HCO(3)(-) exchanger AE2, the Na(+)/H(+) exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H(+)-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at approximately 160 kD for AE2, at approximately 90 kD and approximately 103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl(-) uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO(3)(-) secretion. The molecular identity of the ductal Cl(-)/HCO(3)(-) exchanger remains unclear.
Subject(s)
Anion Transport Proteins , Antiporters , Membrane Proteins/metabolism , Pancreas/metabolism , Proton-Translocating ATPases/metabolism , Sodium-Hydrogen Exchangers/metabolism , Vacuolar Proton-Translocating ATPases , Animals , Cryoultramicrotomy , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Immunoblotting , Immunoenzyme Techniques , Kidney/metabolism , Pancreas/ultrastructure , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , SLC4A Proteins , Sodium-Hydrogen Exchangers/geneticsABSTRACT
Small conductance calcium-gated K(+) channels (SK channels) are encoded by the three SK genes, SK1, SK2, and SK3. These channels likely contribute to slow synaptic afterhyperpolarizations of apamin-sensitive and apamin-insensitive types. SK channels are also widely expressed outside the nervous system. The mouse SK1 gene comprises at least 12 exons extending across 19.8 kb of genomic DNA. This gene encodes a complex pattern of alternatively spliced SK1 transcripts widely expressed among mouse tissues. These transcripts exhibit at least four distinct 5'-nucleotide sequence variants encoding at least two N-terminal amino acid sequences. Optional inclusion of exons 7 and 9, together with two alternate splice donor sites in exon 8, yields transcripts encoding eight variant C-terminal amino acid sequences for SK1. These include an altered putative S6 transmembrane span, modification of the C-terminal cytoplasmic domain binding site for calmodulin, and generation of two alternate predicted binding sites for PDZ domain-containing proteins. 20 of the 32 predicted mouse SK1 transcripts are expressed in brain at levels sufficient to allow consistent detection, and encode 16 SK1 polypeptide variants. Only four of these 16 polypeptides preserve the ability to bind calmodulin in a Ca(2+)-independent manner. Mouse SK1 also exhibits novel, strain-specific, length polymorphism of a polyglutamate repeat in the N-terminal cytoplasmic domain. The evolutionary conservation of this complex transcription pattern suggests a possible role in the tuning of SK1 channel function.