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 Ca2+ in a dose-dependent manner, with a K-1/2 of 230 nM. Thus, Ca2+ 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.