A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.

Jason Tien,Yuh Nung Jan,Huanghe Yang,Xiu Ming Wong,Lily Yeh Jan,Tong Cheng,Christian J Peters

doi:10.7554/elife.02772

Abstract

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility.

Highlights

IntroductionTransmembrane proteins of the TMEM16 family include a number of calcium-dependent ion channels, such as the TMEM16A (Caputo et al, 2008; Schroeder et al, 2008; Yang et al, 2008b) and TMEM16B (Schroeder et al, 2008; Stöhr et al, 2009) calcium-activated chloride channels (CaCCs) and the TMEM16F (Yang et al, 2012) small conductance calcium-activated nonselective cation (SCAN) channel
We began our study of TMEM16A-calcium-activated chloride channels (CaCCs)'s calcium-dependent activation by testing whether CaM is required for channel activity
To test whether the apparent calcium sensitivities of E698Q and E701Q mutant channels are reduced, we measured their steady-state EC50 of calcium-dependent channel activation (Figure 2A,B). We found that their EC50s were shifted to much higher calcium concentrations compared to the EC50 of wildtype TMEM16A channels, suggesting that these two acidic residues might be important for a direct interaction between calcium ions and TMEM16A-CaCC

Summary

Introduction

Transmembrane proteins of the TMEM16 family include a number of calcium-dependent ion channels, such as the TMEM16A (Caputo et al, 2008; Schroeder et al, 2008; Yang et al, 2008b) and TMEM16B (Schroeder et al, 2008; Stöhr et al, 2009) calcium-activated chloride channels (CaCCs) and the TMEM16F (Yang et al, 2012) small conductance calcium-activated nonselective cation (SCAN) channel These proteins have been implicated in a wide range of physiological activities (Hartzell et al, 2009; Kunzelmann et al, 2011; Berg et al, 2012; Scudieri et al, 2012; Huang et al, 2012a) including the control of fluid secretion in epithelia (Romanenko et al, 2010; Huang et al, 2012b), the regulation of membrane excitability in neurons (Billig et al, 2011; Huang et al, 2012c), the scrambling of membrane lipids in platelets (Suzuki et al, 2010; Yang et al, 2012), and the metastasis of certain cancers (Katoh, 2003; Duvvuri et al, 2012).

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