Cl- channels in basolateral renal medullary membranes, VI. Cl- conductance expression in Xenopus oocytes

1992 ◽  
Vol 263 (5) ◽  
pp. F979-F984 ◽  
Author(s):  
L. Zimniak ◽  
W. B. Reeves ◽  
T. E. Andreoli
Keyword(s):  
Gradient Centrifugation ◽  
Membrane Conductance ◽  
Initial Step ◽  
Rabbit Kidney ◽  
Xenopus Laevis Oocytes ◽  
Sequence Information ◽  
Thick Ascending Limb ◽  
Cl Channel ◽  
Cl Channels ◽  
Cl Conductance

Cl- channels from basolateral membranes of the mammalian thick ascending limb of Henle differ significantly, in certain of their functional characteristics, from Cl- channels in apical membranes of secretory epithelia such as trachea or small intestine and from certain Cl- channels in the central nervous system. Yet there is no sequence information available about basolateral thick ascending limb Cl- channels. As an initial step in the isolation of a Cl- channel from the thick ascending limb of Henle's loop, we attempted a functional expression of a Cl- conductance in oocytes from Xenopus laevis. Oocytes injected with mRNA isolated from the outer medulla of rabbit kidney had a membrane conductance, measured using a two-electrode voltage clamp, which was sixfold greater than in water-injected oocytes (9.05 +/- 1.56 vs. 1.43 +/- 0.15 microS, respectively). Ion substitution experiments showed the conductance in the RNA-injected oocytes to be Cl- selective. In addition, the Cl(-)-channel blocker diphenylamine-2-carboxylate (1 mM) almost completely inhibited (79 +/- 6%) the increased conductance seen in the RNA-injected oocytes. Fractionation of the mRNA by sucrose gradient centrifugation revealed that peak Cl- channel activity was expressed using an mRNA fraction of 1.8–3.2 kb in size. These results demonstrate that a membrane Cl- conductance can be expressed in X. laevis oocytes injected with size-selected fractions of mRNA from rabbit outer renal medulla.

cAMP-activated C1 conductance is expressed in Xenopus oocytes by injection of shark rectal gland mRNA

1991 ◽  
Vol 260 (3) ◽  
pp. C664-C669 ◽  
Author(s):  
S. K. Sullivan ◽  
K. Swamy ◽  
M. Field
Keyword(s):  
Xenopus Oocytes ◽  
Functional Characterization ◽  
Equilibrium Potential ◽  
Rectal Gland ◽  
Calcium Buffer ◽  
Cl Channel ◽  
Shark Rectal Gland ◽  
Cl Channels ◽  
Cl Conductance

Development of reliable expression systems for use in identification and functional characterization of proteins required for secretory Cl channel activity is key to understanding the molecular basis of cystic fibrosis (CF). Until now, heterologous expression of epithelial Cl channels had not been accomplished. We show here that Xenopus oocytes express an adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl conductance after injection of mRNA from shark rectal gland. Current through this conductance was rapidly activated by intracellular application of cAMP, reversed near the chloride equilibrium potential (ECl), blocked by the Cl channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoate, and was not affected by preincubation with the intracellular calcium buffer bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, a condition that prohibits activation of the endogenous Ca-activated Cl conductance.

Psoralens: novel modulators of Cl- secretion

1997 ◽  
Vol 272 (3) ◽  
pp. C976-C988 ◽  
Author(s):  
D. C. Devor ◽  
A. K. Singh ◽  
R. J. Bridges ◽  
R. A. Frizzell
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Receptor Activation ◽  
Sustained Response ◽  
Rat Colon ◽  
Cl Secretion ◽  
Cl Channel ◽  
Cl Channels ◽  
Cl Conductance

We evaluated effects of psoralens on Cl- secretion (short-circuit current, I(sc)) across T84 monolayers. Methoxsalen failed to increase I(sc). Several observations suggest that psoralens open cystic fibrosis transmembrane conductance regulator Cl- channels. 1) After activation of the Ca2+-dependent basolateral membrane K+ channel (K(Ca)) by 1-ethyl-2-benzimidazolinone or thapsigargin, methoxsalen (10 microM) further increased I(sc). 2) When added before carbachol (CCh), methoxsalen potentiated the I(sc) response to CCh, as predicted, if it increased apical Cl- conductance. 3) After establishment of a mucosal-to-serosal Cl- gradient and permeabilization of basolateral membrane with nystatin, psoralens increased Cl- current, which was inhibited by glibenclamide. In contrast, neither TS-TM calix[4]arene nor Cd2+, inhibitors of outwardly rectifying Cl- channels and the ClC-2 Cl-channel, respectively, inhibited psoralen-induced Cl- current. In contrast to their effects on Cl- conductance, psoralens failed to significantly affect basolateral membrane K+ conductance; subsequent addition of 1-ethyl-2-benzimidazolinone induced a large increase in K+ conductance. Also, in excised patches, methoxsalen failed to activate K(Ca). In addition to potentiating the peak response to CCh, psoralens induced a secondary, sustained response. Indeed, when added up to 60 min after return of CCh-induced I(sc) to baseline, psoralens induced a sustained I(sc). This sustained response was inhibited by atropine, demonstrating the requirement for continuous muscarinic receptor activation by CCh. This sustained response was inhibited also by verapamil, removal of bath Ca2+, and charybdotoxin. These results suggest that return of I(sc) to baseline after CCh stimulation is not due to downregulation of Ca2+ influx or K(Ca). Finally, we obtained similar results with psoralens in rat colon and primary cultures of murine tracheal epithelium. On the basis of these observations, we conclude that psoralens represent a novel class of Cl- channel openers that can be used to probe mechanisms underlying Ca2+-mediated Cl- secretion.

scholarly journals Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function

2021 ◽  
Author(s):  
Qianyi Wu ◽  
Azman Akhter ◽  
Shashank Pant ◽  
Eunjoo Cho ◽  
Jin Xin Zhu ◽  
...  
Keyword(s):  
Glial Cells ◽  
Excitatory Amino Acid ◽  
Physiological Role ◽  
Glutamate Transport ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Channel Activity ◽  
Excitatory Neurotransmitter ◽  
Cl Channel ◽  
Cl Channels

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory Amino Acid Transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of five additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations with decreased hEAAT1 Cl- channel activity and functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. We also identified a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels

1996 ◽  
Vol 270 (6) ◽  
pp. F1066-F1072 ◽  
Author(s):  
L. Zimniak ◽  
C. J. Winters ◽  
W. B. Reeves ◽  
T. E. Andreoli
Keyword(s):  
Lipid Bilayers ◽  
Antisense Oligonucleotide ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Western Blots ◽  
Cl Channel ◽  
Medullary Thick Ascending Limb ◽  
Cl Channels

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.

Cl− channels in basolateral renal medullary membranes XII. Anti-rbClC-Ka antibody blocks MTAL Cl−channels

1997 ◽  
Vol 273 (6) ◽  
pp. F1030-F1038 ◽  
Author(s):  
Christopher J. Winters ◽  
Ludwika Zimniak ◽  
W. Brian Reeves ◽  
Thomas E. Andreoli
Keyword(s):  
Lipid Bilayers ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Medullary Thick Ascending Limb ◽  
Acid Fragment ◽  
Cl Channels

Cl− channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl−concentrations. rbClC-Ka, a ClC Cl− channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl− transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl− uptake and efflux were measured in suspensions of mouse MTAL segments. Cl− uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3′,5-cyclic monophosphate (DBcAMP). Cl− efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 ± 20 to 196 ± 36 nmol ⋅ mg protein−1 ⋅ 45 s−1( P = 0.003). Cl− efflux was inhibited by the Cl− channel blocker diphenylamine-2-carboxylate (154 ± 26 vs. 70 ± 21 nmol ⋅ mg protein−1 ⋅ 45 s−1, P = 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl− channels in lipid bilayers, reduced Cl− efflux from intact MTAL segments (154 ± 28 vs. 53 ± 14 nmol ⋅ mg protein−1 ⋅ 45 s−1, P = 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl− channel that mediates vasopressin-stimulated net Cl− transport in the MTAL segment.

Chloride channels in apical and basolateral membranes of CCD cells (RCCT-28A) in culture

1992 ◽  
Vol 263 (2) ◽  
pp. F243-F250 ◽  
Author(s):  
P. Dietl ◽  
B. A. Stanton
Keyword(s):  
Chloride Channels ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Cl Channel ◽  
Cl Channels ◽  
Inside Out ◽  
Cl Conductance

Previously, we found that isoproterenol activates whole cell Cl- conductance by a pathway involving adenosine 3',5'-cyclic monophosphate and protein kinase A (PKA) in a renal cell line (RCCT-28A) derived from the cortical collecting duct. The goal of the present study was to determine whether PKA activates Cl- channels in the apical and/or basolateral membrane. Using the patch clamp technique we found a 305-pS Cl- channel, described previously (22), located exclusively in the apical membrane and an outwardly rectifying Cl- channel (13/96 pS) located exclusively in the basolateral membrane. The outward rectifier was highly selective to Cl- versus cations, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, but was not regulated by cytoplasmic pH or Ca2+. Neither isoproterenol nor PKA activated the 305-pS Cl- channel. In contrast, PKA activated a subset of outwardly rectifying channels in inside-out patches. In another subset of outwardly rectifying channels, formation of the inside-out configuration increased channel activity. These channels, however, were not sensitive to PKA. In conclusion, these experiments show that isoproterenol increases the Cl- conductance of RCCT-28A cells by activating a subset of outwardly rectifying Cl- channels located in the basolateral membrane.

scholarly journals Primary structure of a novel 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS)-binding membrane protein highly expressed in Torpedo californica electroplax

1989 ◽  
Vol 261 (1) ◽  
pp. 155-166 ◽  
Author(s):  
T J Jentsch ◽  
A M Garcia ◽  
H F Lodish
Keyword(s):  
Membrane Protein ◽  
Electric Organ ◽  
Membrane Conductance ◽  
Xenopus Laevis Oocytes ◽  
Western Blots ◽  
Torpedo Californica ◽  
Cl Channel ◽  
Functional Part ◽  
Disulphonic Acid ◽  
Membrane Spanning

Polyclonal rabbit antibodies were raised against 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS), an inhibitor of a variety of anion transport proteins. These antibodies specifically recognize SITS-reacted erythrocyte band 3 in immunoprecipitations and Western blots. In Western blots of SITS-reacted membrane proteins derived from vesicles of the electric organ of Torpedo californica (known to express a SITS-sensitive Cl- channel) the antibodies recognized two major species of approximately 93 kDa and approximately 105 kDa. The approximately 93 kDa protein was identified as the alpha-subunit of the Na,K-ATPase. The approximately 105 kDa protein (designated sp105) is a glycoprotein which binds to wheat-germ agglutinin and concanavalin A and is present as a disulphide-linked homodimer under non-reducing conditions. A partial amino acid sequence and a polyclonal antibody were used to clone the corresponding cDNA. sp105 is encoded in electroplax by two abundant mRNAs of approximately 6 and approximately 6.8 kb. A hybridizing mRNA of approximately 5 kb was over 200-fold and over 500-fold less abundant in brain and heart respectively. Sequence analysis of the cDNA predicted a novel protein of 697 amino acids containing eight potential N-linked glycosylation sites. Analysis of hydrophobicity indicated the presence of at least one, and possibly three, putative membrane-spanning domains. When expressed from the Sp6 message in Xenopus laevis oocytes, the protein was inserted into membranes, glycosylated and processed to form a dimer. However, no increase in 36Cl uptake or in membrane conductance could be detected. We found no effect of hybrid depleting the specific message on expression of the Torpedo electroplax Cl- channel in oocytes. Thus we conclude that this novel electroplax membrane protein is probably not a functional part of the chloride channel.

Carbonic anhydrase XII mRNA encodes a hydratase that is differentially expressed along the rabbit nephron

2003 ◽  
Vol 284 (2) ◽  
pp. F399-F410 ◽  
Author(s):  
George J. Schwartz ◽  
Anne M. Kittelberger ◽  
Richard H. Watkins ◽  
Michael A. O'Reilly
Keyword(s):  
Carbonic Anhydrase ◽  
Transmembrane Protein ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Thick Ascending Limb ◽  
Basolateral Membranes ◽  
Collecting Ducts ◽  
Straight Tubules ◽  
Hydratase Activity

Membrane-bound carbonic anhydrase (CA) facilitates acidification in the kidney. Although most hydratase activity is considered due to CA IV, some in the basolateral membranes could be attributed to CA XII. Indeed, CA IV is glycosylphosphatidylinositol anchored, connoting apical polarization, but CA IV immunoreactivity has been detected on basolateral membranes of proximal tubules. Herein, we determined whether CA XII mRNA was expressed in acidifying segments of the rabbit nephron. The open reading frame of CA XII was sequenced from a rabbit kidney cortex cDNA library; it was 83% identical to human CA XII and coded for a 355-amino acid single-pass transmembrane protein. Northern blot analysis revealed an abundant 4.5-kb message in kidney cortex, medulla, and colon. By in situ hybridization, CA XII mRNA was expressed by proximal convoluted and straight tubules, cortical and medullary collecting ducts, and papillary epithelium. By RT-PCR, CA XII mRNA was abundantly expressed in cortical and medullary collecting ducts and thick ascending limb of Henle's loop; it was also expressed in proximal convoluted and straight tubules but not in glomeruli or S3 segments. FLAG-CA XII of ∼40 kDa expressed in Escherichia coli showed hydratase activity that was inhibited by 0.1 mM acetazolamide. Unlike CA IV, expressed CA XII activity was inhibited by 1% SDS, suggesting insufficient disulfide linkages to stabilize the molecule. Western blotting of expressed CA XII with two anti-rabbit CA IV peptide antibodies showed no cross-reactivity. Our findings indicate that CA XII may contribute to the membrane CA activity of proximal tubules and collecting ducts.

Stilbene disulfonate blockade of colonic secretory Cl- channels in planar lipid bilayers

1989 ◽  
Vol 256 (4) ◽  
pp. C902-C912 ◽  
Author(s):  
R. J. Bridges ◽  
R. T. Worrell ◽  
R. A. Frizzell ◽  
D. J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Kinetic Scheme ◽  
Disulfonic Acid ◽  
Lipid Bilayer Membranes ◽  
Plasma Membrane Vesicles ◽  
Open Probability ◽  
Cl Channel ◽  
Cl Channels

We studied blockade by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) of a secretory Cl- channel from colonic enterocyte plasma membrane vesicles incorporated into planar lipid bilayer membranes. Except for intermittent long-lived closed periods (100 ms to several min), the control channel open probability (Po) was greater than 90%. DNDS, added to the cis or vesicle-containing side, which corresponds to the outer membrane side of the channel, caused a dramatic increase in the number of current transitions from the open-to-closed state. DNDS caused a concentration-dependent decrease in Po with a maximum inhibition of 95 +/- 2.0% and a half-maximal inhibitory concentration of 3.3 +/- 1.4 microM. DNDS added to the trans side of the channel had no effect on either the single-channel conductance or kinetic behavior of the channel. Kinetic analysis revealed that DNDS blockade from the cis side could be explained by a linear, closed-open-blocked, kinetic scheme. The estimated DNDS block rate constants were kon = 3.2 X 10(7) M-1.s-1 and koff = 52 s-1, yielding an equilibrium dissociation constant (KD) of 2.1 +/- 0.38 microM, similar to the Ki for inhibition of Po. The effects of DNDS were fully reversible after perfusion of the cis compartment with DNDS-free solution. In contrast, the covalently reactive 4,4'-diisothiocyano-substituted stilbene disulfonate caused an irreversible blockade of the Cl- channel.

scholarly journals Volume-activated chloride currents are not correlated with P-glycoprotein expression

1995 ◽  
Vol 307 (3) ◽  
pp. 713-718 ◽  
Author(s):  
C De Greef ◽  
J Sehrer ◽  
F Viana ◽  
K van Acker ◽  
J Eggermont ◽  
...  
Keyword(s):  
Cell Types ◽  
Protein Product ◽  
Chloride Currents ◽  
Cl Channel ◽  
P Glycoprotein ◽  
Hela Cell Lines ◽  
Mdr1 Mrna ◽  
Order Of Magnitude ◽  
Mdr1 Expression ◽  
Cl Channels

It has been proposed that P-glycoprotein, the product of the human MDR1 gene, may function not only as a drug transporter but, depending on the conditions, as a volume-activated Cl- channel [Valverde, Diaz, Sepúlveda, Gill, Hyde and Higgins (1992) Nature (London) 355, 830-833; Gill, Hyde, Higgins, Valverde, Mintenig and Sepúlveda (1992) Cell 71, 23-32]. To verify this hypothesis, we have compared volume-activated Cl- currents with the level of MDR1 mRNA and its protein product in the human KB3 (epitheloid lung cancer) and HeLa cell lines. The related MDR2 was also included to find out whether it could account for observed discrepancies between Cl- current and MDR1 expression. A 40% decrease in osmolarity evoked a Cl- current in both cell types (at +80 mV: 50.3 +/- 4.3 pA/pF in KB3, n = 13; 28.2 +/- 3.3 pA/pF in HeLa, n = 16). The blocking of this current in both cell types by 5-nitro-2-(3-phenylpropylamino)-benzoic acid and by 1,9-dideoxyforskolin is similar to that of the presumed P-glycoprotein associated Cl- channel. As measured by reverse-transcriptase polymerase chain reaction, KB3 cells expressed only an extremely small amount of the messengers for MDR1 and MDR2. The signal observed for MDR1 in HeLa cells was at least an order of magnitude more intense than in KB3 cells, while MDR2 mRNA was undetectable. A clear difference in MDR1 expression between KB3 and HeLa was also observed at the protein level. These data are difficult to reconcile with the hypothesis that in HeLa and KB3 cells MDR1- or MDR2- encoded P-glycoproteins are associated with volume-activated Cl- channels.

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