Characterization of Cytosolic Cl&lt;sup&gt;&amp;ndash;&lt;/sup&gt; Channel Inhibitors by Size Exclusion Chromatography

Krick, Wolfgang; Disser, J.; Rabe, Alexander; Frömter, Eberhard; Hansen, Ch. P.; Roch, Brigitte; Kunzelmann, Karl; Greger, R.; Fehlhaber, H.‐W.; Burckhardt, G.

doi:10.1159/000154761

Cited by 6 publications

(10 citation statements)

References 11 publications

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“…Based on its spontaneous activation after excision we have speculated previously that the channel in intact cells probably is blocked by a cytosolic inhibitor that diffuses away after excision. Subsequently, and in collaboration with others [11,12,14], we have succeeded in demonstrating the existence of such an inhibitory principle in cytoplasm. It is presumably a low-molecular weight (approximately.…”

Section: Introductionmentioning

confidence: 63%

Micromolar concentrations of steroids and of aldosterone antagonists inhibit the outwardly rectifying chloride channel with different kinetics

Rabe

Frömter

2000

Pflügers Arch – Eur J Physiol

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We used the patch-clamp technique to analyse the open/close kinetics of single, outwardly rectifying, intermediate-conductance (ORIC) Cl- channels from cultured epithelial cells under control conditions and in presence of different inhibitors. As observed previously in excised inside/out patches under control conditions, the switching kinetics were characterized by one open-state time constant (tau0 is approximately 30 ms) and three closed-state time constants (tau(cl)is approximately = to 0.2 ms, tau(c2) is approximately = 2 ms and tau(c3) is approximately = 60 ms). Aldosterone, six further steroids and two aldosterone antagonists inhibited channel open probability (NPo) concentration dependently with the potency at 10 micromol/l increasing in the sequence: hydrocortisone, corticosterone, P-oestradiol, cortisone, aldosterone, testosterone, progesterone, canrenone, spironolactone. Although all substances decreased tau(o), neither the steroids nor the aldosterone antagonists affected tau(cl), tau(c2) or tau(c3) or induced additional transitions with additional time constants. Instead, the steroids increased the prevalence of tau(c2) in the dwell-time histograms and the aldosterone antagonists increased the prevalence of tau(c3), both in a concentration-dependent manner. These observations may be explained by a model in which one open state leads to one of three closed states with rate constants alpha, beta and gamma, and in which beta or gamma increase under the influence of steroids or aldosterone antagonists, respectively. Cytosol, which contains a Cl- channel inhibitor of unknown molecular structure, (Krick et al., Pflügers Arch 418:491, 1991) was also tested, but the results did not conform to the blocker mechanisms described above. This shows that there are even further modes of channel inhibition and argues against the cytosolic Cl- channel inhibitor being a steroid.

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Section: Introductionmentioning

confidence: 63%

Micromolar concentrations of steroids and of aldosterone antagonists inhibit the outwardly rectifying chloride channel with different kinetics

Rabe

Frömter

2000

Pflügers Arch – Eur J Physiol

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“…Furthermore, ICOR-type channels are observed after excision from normal or CF cells equally frequently [48]. In a collaborative effort, we have attempted to identify a cytosolic inhibitor preventing the activation of the ICOR channel on the cell [44,45,49]. At this stage it seems likely that the inhibitor is an intermediate size (ca.…”

Section: The Icor Channelmentioning

confidence: 99%

The membrane transporters regulating epithelial NaCl secretion

Greger

1996

Pflügers Arch.

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Ten years ago, the basic principles operating in one specific, albeit non-mammalian, exocrine gland, the rectal gland of Squalus acanthias, were described in detail. The concept emerging from these studies appeared applicable to almost any other exocrine gland, because it involved membrane transporters which are also present in mammalian epithelial cells. Meanwhile, it has become clear that the mechanisms of NaCl secretion are diverse: the mechanisms of NaCl uptake; the ion channels involved; and also the mechanisms of hormonal control. Nevertheless, several steps in NaCl secretion still appear to be uniform: (1) several signalling pathways converge and act cooperatively, (2) one primary regulatory step is the upregulation of the luminal Cl- conductance, (3) secondarily active NaCl uptake mechanisms are upregulated, (4) increasing evidence links NaCl secretion to membrane trafficking and (5) the entire machinery seems to be primed to secure cellular homeostasis in terms of cytosolic ion concentrations. This brief review summarizes the mechanisms of control of NaCl secretion. The major issues addressed are the NaCl uptake mechanisms, the ion channels involved and the cellular mechanisms coordinating secretion. The major NaCl secreting cells discussed here will be the respiratory epithelial cells, the exocrine cells of pancreatic acini and the cells of colonic crypts.

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“…The properties of the anion conductance in vesicles resemble more those of the ICOR/ORCC, although considerably higher concentrations of NPPB, DIDS and glibenclamide were required for half-maximal inhibition of conductive 36 Cl -uptake than of Cl -currents in single channel experiments. Like the ICOR/ORCC [16,20,24], the Cl -conductance can be inhibited by intravesicular trapping of cytosol (preliminary data not shown) indicating that vesicles from HT29 cells may prove valuable in the search for the cytosolic Cl -channel inhibitors [21].…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, cytosol from a variety of sources reversibly and dose dependently inhibits the ICOR/ORCC in excised patches [16,20,21,24]. The putative inhibitor(s) is (are) resistant to hydrolases and has a molecular mass of 700-1,500 Da [24].…”

Section: Introductionmentioning

confidence: 99%

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Chloride conductance in HT29 cells: investigations with apical membrane vesicles and RT-PCR

Hagos¹,

Krick²,

Burckhardt³

1999

Pfl�gers Archiv European Journal of Physiology

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Vesicles enriched in a marker enzyme for apical membranes were isolated from HT29 cells. These vesicles contain an anion conductance with the selectivity gluconate approximately sulphate4, 4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS)>glibenclamide. The Cl- conductance was insensitive to Ca2+ and to extravesicular cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inosine 5'-triphosphate (ITP). Using the reverse transcription polymerase chain reaction (RT-PCR) technique and sequencing of the amplified products we detected messenger ribonucleic acid (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR), the putative Cl- channel or Cl- channel regulator pICln, and the Cl- channels ClC-2, ClC-3, ClC-5 and ClC-6 in HT29 cells. The properties of the vesicles' Cl- conductance resemble those of the intermediate conductance outwardly rectifying Cl- channel and tentatively exclude contributions of CFTR, pICln and ClC-2. Whether ClC-3, ClC-5, ClC-6 are involved in Cl- conductance remains to be determined.

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Characterization of Cytosolic Cl<sup>–</sup> Channel Inhibitors by Size Exclusion Chromatography

Cited by 6 publications

References 11 publications

Micromolar concentrations of steroids and of aldosterone antagonists inhibit the outwardly rectifying chloride channel with different kinetics

Micromolar concentrations of steroids and of aldosterone antagonists inhibit the outwardly rectifying chloride channel with different kinetics

The membrane transporters regulating epithelial NaCl secretion

Chloride conductance in HT29 cells: investigations with apical membrane vesicles and RT-PCR

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