Ca²⁺-activated Cl^- current from human bestrophin-4 in excised membrane patches

Bestrophins are a newly discovered family of Cl^- channels, some members of which are activated by intracellular Ca²⁺. So far, all studies were carried out with whole-cell recordings from plasmid-transfected cultured cells, so it is unclear whether Ca²⁺ activates bestrophin through a metabolic mechanism or in a more direct way. We report here experiments that addressed this question with excised, inside-out membrane patches. We chose human bestrophin-4 (hBest4) for heterologous expression because it gave particularly large Cl^- currents when expressed, thus allowing detection even in excised membrane patches. hBest4 gave a negligible Cl^- current in a Ca²⁺-free solution on the cytoplasmic (bath) side, but produced a Cl^- current that was activated by Ca ²⁺ in a dose-dependent manner, with a K1/2 of 230 nM. Thus, Ca ²⁺ appears to activate the bestrophin Cl^- channel without going through a freely diffusible messenger or through protein phosphorylation. Because the activation and deactivation kinetics were very slow, however, we cannot exclude the involvement of a membrane-associated messenger.