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.

Functional identification of a sarcolemmal chloride channel from bovine tracheal smooth muscle

1996 ◽  
Vol 271 (5) ◽  
pp. C1716-C1724 ◽  
Author(s):  
D. Salvail ◽  
A. Alioua ◽  
E. Rousseau
Keyword(s):  
Smooth Muscle ◽  
Lipid Bilayers ◽  
Reversal Potential ◽  
Tracheal Smooth Muscle ◽  
Disulfonic Acid ◽  
Equilibrium Potential ◽  
Functional Identification ◽  
Cl Channels ◽  
Cl Conductance

The biophysical and pharmacological characteristics of unitary Cl- currents from bovine tracheal smooth muscle cells were studied after reconstitution of microsomal vesicles into planar lipid bilayers. Two types of currents were recorded simultaneously in KCl buffer: the well-defined Ca(2+)-dependent K+ conductance [GK(Ca)] and a much smaller Cl- current, indicating that the Cl- channels under scrutiny originate from the same membrane as the GK(Ca)-type channels, the plasma membrane of airway smooth muscle (ASM) cells. The GK(Ca) activities were eliminated by the use of CsCl buffer. The average unitary Cl- conductance measured in 50 mM trans-250 mM cis CsCl was 77 +/- 6 pS (n = 21), and the reversal potential measured in various CsCl gradients followed the Cl- equilibrium potential as determined from the Nernst equation. In contrast with the previous reports describing the Ca2+ sensitivity of macroscopic ASM Cl- currents, this channel was found to be insensitive to cytoplasmic and extracellular Ca2+ levels. Phosphorylation cocktails, including protein kinases A, G, or C, did not alter the activity of the channel nor did changes in pH. Among a series of Cl- channel inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid [50% effective concentration (EC50) = 30 microM] and 5-nitro-2-(3-phenylpropylamino) benzoic acid (EC50 = 130 microM) were the most potent blockers of the current examined. The exact role of this surface Cl- conductance remains unclear, and its involvement in cellular activity needs further investigation.

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.

Functional Characterization of a ClC-2-Like Cl− Conductance in Surface Epithelial Cells of Rat Rectal Colon

2010 ◽  
Vol 235 (1) ◽  
pp. 27-41 ◽  
Author(s):  
Akihiro Inagaki ◽  
Soichiro Yamaguchi ◽  
Hiromi Takahashi-Iwanaga ◽  
Toshihiko Iwanaga ◽  
Toru Ishikawa
Keyword(s):  
Epithelial Cells ◽  
Functional Characterization ◽  
Cl Conductance ◽  
Rectal Colon

Cl- secretion by cultured shark rectal gland cells. II. Effects of forskolin on cellular electrophysiology

1991 ◽  
Vol 260 (4) ◽  
pp. C824-C831 ◽  
Author(s):  
W. M. Moran ◽  
J. D. Valentich
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Ringer Solution ◽  
Rectal Gland ◽  
Electrical Potential Difference ◽  
Electrophysiological Properties ◽  
Shark Rectal Gland ◽  
Membrane Electrical Potential ◽  
Cl Conductance

Employing microelectrode techniques we have assessed the cellular electrophysiological properties of shark rectal gland (SRG) cells in primary culture. In the absence of secretagogues a 10-fold reduction in the Cl- concentration of the apical superfusate shark Ringer solution had little effect on either apical membrane electrical potential difference (Va) or fractional resistance (fRa), indicating little, if any, apical membrane Cl- conductance. Superfusing the basolateral surface with high-K+ shark Ringer solution (K+ increased 10-fold) depolarized the basolateral membrane electrical potential difference (Vb) by 43 mV, indicating that this barrier is largely K+ conductive. In addition, basolateral Ba2+ (5 mM) depolarized Vb by 12 mV and reduced fRa from 0.92 to 0.58, results consistent with a K(+)-conductive basolateral membrane in unstimulated SRG cells. Basolateral forskolin (10(-6) M) depolarized Va by 25 mV and caused a dramatic reduction in fRa from 0.97 to approximately 0.10. Under these conditions, a 10-fold decrease in apical superfusate Cl- concentration depolarized Va by 37 mV, revealing an adenosine 3',5'-cyclic monophosphate-induced apical membrane Cl- conductance. The time course of the forskolin-induced changes in Va and Vb suggests that the basolateral membrane K+ conductance increased and maintained the driving force for apical Cl- exit, as in other Cl(-)-secreting epithelia. These electrophysiological properties compare favorably with those of the perfused SRG tubule and indicate that SRG primary cultures are a suitable model for Cl(-)-secreting epithelia.

scholarly journals Functional Characterization of the Arabidopsis Abscisic Acid Transporters NPF4.5 and NPF4.6 in Xenopus Oocytes

2020 ◽  
Vol 11 ◽  
Author(s):  
Sophie Léran ◽  
Mélanie Noguero ◽  
Claire Corratgé-Faillie ◽  
Yann Boursiac ◽  
Chantal Brachet ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Xenopus Oocytes ◽  
Functional Characterization

scholarly journals Revisiting the functional properties of NPF6.3/NRT1.1/CHL1 in xenopus oocytes

2018 ◽  
Author(s):  
Mélanie Noguero ◽  
Sophie Léran ◽  
Eléonore Bouguyon ◽  
Chantal Brachet ◽  
Pascal Tillard ◽  
...  
Keyword(s):  
Transport Properties ◽  
Xenopus Oocytes ◽  
Nitrate Concentration ◽  
Functional Characterization ◽  
Nitrate Transport ◽  
Experimental Conditions ◽  
Point Mutant ◽  
Electrode Voltage ◽  
Nitrate Transporters

ABSTRACTWithin the Arabidopsis NPF proteins, most of the characterized nitrate transporters are low-affinity transporters, whereas the functional characterization of NPF6.3/NRT1.1 has revealed interesting transport properties: the transport of nitrate and auxin, the eletrogenicity of the nitrate transport and a dual-affinity transport behavior for nitrate depending on external nitrate concentration. However, some of these properties remained controversial and were challenged here. We functionally express WT NPF6.3/NRT1.1 and some of its mutant in Xenopus oocytes and used a combination of uptake experiments using 15N-labelled nitrate and two-electrode voltage-clamp. In our experimental conditions in xenopus oocytes, in the presence or in the absence of external chloride, NPF6.3/NRT1.1 behaves as a non-electrogenic and pure low-affinity transporter. Moreover, further functional characterization of a NPF6.3/NRT1.1 point mutant, P492L, allowed us to hypothesize that NPF6.3/NRT1.1 is regulated by internal nitrate concentration and that the internal perception site involves the P492 residue.

Characterization of the effects of Cl− channel modulators on TMEM16A and bestrophin-1 Ca2+ activated Cl− channels

2014 ◽  
Vol 467 (7) ◽  
pp. 1417-1430 ◽  
Author(s):  
Yani Liu ◽  
Huiran Zhang ◽  
Dongyang Huang ◽  
Jinlong Qi ◽  
Jiaxi Xu ◽  
...  
Keyword(s):  
Cl Channel ◽  
Cl Channels

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.

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