Two Ca(2+)-Binding Sites Cooperatively Couple Together in TMEM16A Channel

Area of Science:

• Biophysics
• Molecular Biology
• Ion Channel Physiology

Background:

• TMEM16A functions as a calcium-activated chloride channel.
• Understanding the Ca(2+) gating mechanism of TMEM16A is crucial but remains unclear.
• Ca(2+) sensors dynamically control TMEM16A channel gating.

Purpose of the Study:

• To elucidate the cooperative mechanism of Ca(2+) sensors in TMEM16A.
• To investigate how Ca(2+) binding and channel opening occur.
• To analyze the functional impact of mutations in Ca(2+)-sensitive domains.

Main Methods:

• Site-directed mutagenesis of TMEM16A Ca(2+)-sensitive domains.
• Electrophysiological recordings to measure channel activity.
• Determination of EC50 values for wild-type and mutant channels.

Main Results:

• Two cooperative Ca(2+) sensors were confirmed in TMEM16A.
• Mutations E447Y and E702Q-E705Q significantly weakened Ca(2+) affinity (EC50 increased).
• A triple mutation demonstrated cooperative interaction between the two Ca(2+)-sensitive domains, further reducing Ca(2+) sensitivity.

Conclusions:

• TMEM16A possesses two cooperative Ca(2+) sensors that regulate channel gating.
• Ca(2+) and Sr(2+) utilize a shared gating mechanism for TMEM16A.
• This study provides mechanistic insights into TMEM16A activation by divalent cations.