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Ca2+-independent effects of BAPTA and EGTA on single-channel Cl- currents in brown adipocytes
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
2007 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1768, no 11, 2714-2725 p.Article in journal (Refereed) Published
Abstract [en]

The Cl channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl channels (ORCC). To study tentative Ca2+ regulation of these channels, we attempted to control Ca2+ levels at the cytoplasmic side of the inside-out membrane patches with Ca2+-chelating agents. However, we found that the commonly used Ca2+-chelators EGTA and BAPTA by themselves influenced the Cl channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca2+-chelators (acting from the cytoplasmic side) on the single Cl channel currents in patch-clamp experiments. Using fixed (1–2 mM) concentrations of chelators, two types of Cl channels were identified, as discriminated by their reaction to the Ca2+-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca2+. The anionic classical Cl channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca2+ level control in themselves could fundamentally affect the behaviour of Cl channels.

Place, publisher, year, edition, pages
2007. Vol. 1768, no 11, 2714-2725 p.
Keyword [en]
Chloride channel; Calcium; Calcium chelator; EGTA; BAPTA; Single channel kinetics; DIDS; SITS
National Category
Biological Sciences
URN: urn:nbn:se:su:diva-23753DOI: 10.1016/j.bbamem.2007.07.003ISI: 000251493700007OAI: diva2:194350
Part of urn:nbn:se:su:diva-474Available from: 2005-04-21 Created: 2005-04-21 Last updated: 2010-08-09Bibliographically approved
In thesis
1. Chloride Channels and Brown Fat Cells
Open this publication in new window or tab >>Chloride Channels and Brown Fat Cells
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chloride ion channels are macromolecular pores providing for passage of chloride ions (and certain other inorganic and organic anions) through the cell membrane, down their electrochemical gradients. Chloride channels are differentially expressed in various cells, to best suit specific cellular activities. They are present in practically all living cells, and regardless of cell specialization they play an important role in vital housekeeping functions of cell-volume and pH regulation and in membrane potential stabilization. Regulation of cell volume underlies the structural integrity and constancy of the intracellular milieu. A variety of metabolic pathways have been shown to be sensitive to cell volume, and alterations of cell volume and osmoregulation processes can influence various intracellular signaling and organizing factors.

Volume-regulated anion channels (VRACs) are believed to play a pivotal role in cell-volume regulating processes. In this report I present data from macroscopic patch-clamp studies of VRACs performed in a fibroblast cell line and from single channel studies of chloride channels (tentatively related to VRACs) in mouse brown adipocytes in primary culture.

One of the characteristic features of the VRACs is their dependence on the presence of cytoplasmic ATP. In whole-cell experiments, removal of ATP from the pipette solution almost completely prevented activation of VRACs, whereas substitution of ATP with the nonhydrolyzable analog ATPγS did not alter the activation of VRACs. The inhibitors of protein tyrosine kinases (PTK) tyrphostin A25 and B46 depressed VRAC currents in both cases (ATP and ATPγS), but a PTK ineffective analog (tyrphostin A1) did not affect VRAC currents. We infer that in the cell preparation we used, ATP has a dual role in VRAC regulation: it is required for channel-protein phosphorylation and it can influence channel activity through non-hydrolytic binding in a ligand-receptor manner. It can additionally be suggested that tyrosine-specific protein kinases can be involved in the regulation of VRACs, independently of the effects of ATP. We also studied cell cycle-related changes in activation of VRACs by osmotic swelling of cells chemically arrested at different phases of the cell cycle. We found no significant changes during most of the cell cycle, except short periods before and after mitosis and in the quiescent G0 state.

The single Cl- channels of brown adipocytes resemble in their electrophysiological phenotype outwardly rectifying Cl- channels (ORCCs). We investigated the sensitivity of these channels to intracellular Ca2+. It appeared that the commonly used Ca2+-chelators EGTA and BAPTA could influence the ORCCs currents by themselves, independently of their calcium chelating effects. In some channels, these chelators induced classical flickery-type block of activity, whereas in others there was quasi-blockage, i.e. a peculiar combination of flickery blockage and overall channel activation. The chloride channel blocking agents DIDS and SITS mimicked the true/quasi blockage of EGTA and BAPTA. These phenomena add to the structure-function characteristics of the ORCC molecule. Moderate inhibitory effect of Ca2+ within a physiological range of intracellular concentrations (sub-µM) was also detected; however, the biological relevance of this observation, as well as of these Cl- channels in general, remains to be clarified.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut för experimentell biologi, 2005. 76 p.
Chloride channel, BAPTA, EGTA, Ca
National Category
urn:nbn:se:su:diva-474 (URN)91-7155-044-5 (ISBN)
Public defence
2005-05-12, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00
Available from: 2005-04-21 Created: 2005-04-21Bibliographically approved

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