Ca2+-activated Cl- current from human bestrophin-4 in excised membrane patches
Related Experiment Video
Updated: Aug 8, 2026
Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices
Published on: January 19, 2012
Takashi Tsunenari1, Jeremy Nathans, King-Wai Yau
1Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. tsune@jhmi.edu
Abstract:
Bestrophins are a newly discovered family of Cl(-) channels, some members of which are activated by intracellular Ca(2+). So far, all studies were carried out with whole-cell recordings from plasmid-transfected cultured cells, so it is unclear whether Ca(2+) 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 Ca(2+)-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 K(1/2) 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.
Insights
Calcium (Ca2+) directly activates bestrophin-4 (hBest4) chloride channels. This study used excised membrane patches to show Ca2+ binding, not a metabolic pathway, triggers channel activity.
Area of Science:
- Molecular Biology
- Ion Channel Physiology
Background:
- Bestrophins are a family of chloride (Cl-) channels.
- Some bestrophins are activated by intracellular calcium (Ca2+).
- Previous studies used whole-cell recordings, leaving the activation mechanism unclear.
Purpose of the Study:
- To investigate the directness of Ca2+ activation of bestrophin channels.
- To determine if Ca2+ activates bestrophins via a metabolic or direct mechanism.
Main Methods:
- Utilized excised, inside-out membrane patches for experiments.
- Employed heterologous expression of human bestrophin-4 (hBest4) for large Cl- currents.
- Measured Cl- currents in response to varying Ca2+ concentrations on the cytoplasmic side.
Main Results:
- hBest4 exhibited negligible Cl- current in Ca2+-free solutions.
- Ca2+ dose-dependently activated hBest4 Cl- currents with a K(1/2) of 230 nM.
- Activation and deactivation kinetics were observed to be very slow.
Conclusions:
- Ca2+ directly activates the bestrophin-4 Cl- channel.
- Activation likely occurs without freely diffusible messengers or protein phosphorylation.
- The possibility of a membrane-associated messenger cannot be excluded due to slow kinetics.
