Ca 2+-independent effects of BAPTA and EGTA on single-channel Cl − currents in brown adipocytes

Abstract

The Cl − channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl − channels (ORCC). To study tentative Ca 2+ regulation of these channels, we attempted to control Ca 2+ levels at the cytoplasmic side of the inside-out membrane patches with Ca 2+-chelating agents. However, we found that the commonly used Ca 2+-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 Ca 2+-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 Ca 2+-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 Ca 2+. 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 Ca 2+ level control in themselves could fundamentally affect the behaviour of Cl − channels.