Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

Area of Science:

• Molecular Biology
• Ion Channel Physiology
• Biophysics

Background:

• TMEM16A channels are crucial for cellular chloride transport.
• Understanding the pore structure is key to elucidating channel gating and ion permeation.
• Positively charged residues in ion channel pores often play critical roles in ion selectivity and function.

Purpose of the Study:

• To investigate the functional impact of mutating positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels.
• To determine how these mutations affect channel kinetics, ion selectivity, ion permeability, and sensitivity to channel blockers.

Main Methods:

• Site-directed mutagenesis was used to introduce specific amino acid substitutions (K613E, K628E, K630E, R646E, R761E) in the X. tropicalis TMEM16A channel pore.
• Electrophysiological techniques were employed to measure activation/deactivation kinetics, current density, anion selectivity, and Na(+) permeability.
• The effects of known chloride channel blockers (A-9-C, DIDS, T16inh-A01) on mutant channels were assessed.

Main Results:

• Mutations did not significantly alter activation/deactivation kinetics, except for K613E, which showed reduced current density.
• K628E and R761E mutations altered anion selectivity but not Na(+) permeation.
• K613E, R646E, and the double mutant K613E + R646E affected both anion selectivity and Na(+) permeability.
• Mutations led to altered blockade by A-9-C, DIDS, and T16inh-A01.

Conclusions:

• The results indicate the presence of at least two anion binding sites within the TMEM16A channel pore, located at electrical distances of 0.3 and 0.5.
• These identified binding sites are critical for both anion permeation and the blockade of the channel by specific inhibitors.
• The study provides insights into the structural determinants of ion selectivity and transport mechanisms in TMEM16A channels.