Abstract
TMEM16A is a ligand-gated anion channel that is activated by intracellular Ca2+. This channel comprises two independent pores and closely apposed Ca2+ binding sites that are contained within each subunit of a homodimeric protein. Previously we characterized the influence of positively charged pore-lining residues on anion conduction (Paulino et al., 2017a). Here, we demonstrate the electrostatic control of permeation by the bound calcium ions in mouse TMEM16A using electrophysiology and Poisson-Boltzmann calculations. The currents of constitutively active mutants lose their outward rectification as a function of Ca2+ concentration due to the alleviation of energy barriers for anion conduction. This phenomenon originates from Coulombic interactions between the bound Ca2+ and permeating anions and thus demonstrates that an electrostatic gate imposed by the vacant binding site present in the sterically open pore, is released by Ca2+ binding to enable an otherwise sub-conductive pore to conduct with full capacity.
Highlights
The calcium-activated chloride channel TMEM16A is part of a large family of membrane proteins that encompasses ion channels and lipid scramblases with a common conserved molecular architecture (Brunner et al, 2016; Caputo et al, 2008; Picollo et al, 2015; Schroeder et al, 2008; Terashima et al, 2013; Whitlock and Hartzell, 2017; Yang et al, 2008)
Recent investigations have defined the structural basis for ion permeation and gating in TMEM16A and revealed features that distinguish this ion channel from other homologues working as lipid scramblases (Brunner et al, 2014; Dang et al, 2017; Paulino et al, 2017a; Paulino et al, 2017b)
Our study demonstrates the strong electrostatic influence of bound Ca2+ ions on anion conduction in the Ca2+-activated Cl- channel TMEM16A
Summary
The calcium-activated chloride channel TMEM16A is part of a large family of membrane proteins that encompasses ion channels and lipid scramblases with a common conserved molecular architecture (Brunner et al, 2016; Caputo et al, 2008; Picollo et al, 2015; Schroeder et al, 2008; Terashima et al, 2013; Whitlock and Hartzell, 2017; Yang et al, 2008). Anions access the narrow neck of an hourglass-shaped pore via water-filled vestibules from the extra- and intracellular sides and permeate through the constricted part presumably after shedding most of their coordinating water molecules (Betto et al, 2014; Dang et al, 2017; Ni et al, 2014; Paulino et al, 2017a; Qu and Hartzell, 2000) During this process, the energetic penalty for dehydration is surmounted by positive charges placed on both sides of the neck (Paulino et al, 2017b). These long-range interactions underlie an electrostatic gating mechanism that acts through space and that is synergistic with the opening of a steric gate, allowing an otherwise sub-conductive pore to conduct with full capacity
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