Gating the pore of the calcium-activated chloride channel TMEM16A

Andy K M Lam,Raimund Dutzler,Jan Rheinberger,Cristina Paulino

doi:10.1038/s41467-020-20787-9

Andy K M Lam, Raimund Dutzler + Show 2 more

Open Access

https://doi.org/10.1038/s41467-020-20787-9

Copy DOI

Abstract

The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca2+.

Highlights

The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore
The most prominent Calcium-activated chloride channels (CACC) is formed by TMEM16A, which is expressed in different tissues of the human body[2,3,4]
As revealed in structures obtained by cryo-electron microscopy, the distinction between TMEM16 channels and scramblases is manifested in a conformational difference of α-helices forming the subunit cavity[11]

Summary

Introduction

The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. The helix α4, lining one edge of the open subunit cavity in the lipid scramblase nhTMEM16, has rearranged in TMEM16A to come in contact with α6 on the opposite edge to form an aqueous pore that is for a large part shielded from the membrane This ion conduction pore has an hourglass shape with wide aqueous cavities leading into a narrow neck from both sides of the membrane[12]. Anions are presumably conducted through the narrow neck with most of their coordinating water stripped, a process that is compensated for electrostatically by positively charged residues located at the extra- and intracellular entry of the neck[11] Both pores in the dimeric protein act independently with respect to activation and ion conduction[18,19]. This is followed by presumed additional conformational changes that lead to the opening of a steric gate that was proposed to be located within the narrow neck[12]

Methods

Results

Conclusion