Ca2+-independent effects of BAPTA and EGTA on single-channel Cl- currents in brown adipocytes

摘要

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. (c) 2007 Elsevier B.V. All rights reserved.