Cryo-EM structures of the TMEM16A calcium-activated chloride channel

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A(1-3)
control neuronal signalling, smooth muscle contraction, airway and
exocrine gland secretion, and rhythmic movements of the gastrointestinal
system(4-7). To understand how CaCCs mediate and control anion
permeation to fulfil these physiological functions, knowledge of the
mammalian TMEM16A structure and identification of its pore-lining
residues are essential. TMEM16A forms a dimer with two pores(8,9).
Previous CaCC structural analyses have relied on homology modelling of a
homologue (nhTMEM16) from the fungus Nectria haematococca that functions
primarily as a lipid scramblase(10-12), as well as
subnanometre-resolution electron cryo-microscopy(12). Here we present de
novo atomic structures of the transmembrane domains of mouse TMEM16A in
nanodiscs and in lauryl maltose neopentyl glycol as determined by
single-particle electron cryo-microscopy. These structures reveal the
ion permeation pore and represent different functional states. The
structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved
within each monomer with a constricted pore; this is likely to
correspond to a closed state, because a CaCC with a single Ca2+
occupancy requires membrane depolarization in order to open
(C.J.P.etal., manuscript submitted). The structure in nanodiscs has two
Ca2+ ions per monomer and its pore is in a closed conformation; this
probably reflects channel rundown, which is the gradual loss of channel
activity that follows prolonged CaCC activation in 1 mM Ca2+. Our
mutagenesis and electrophysiological studies, prompted by analyses of
the structures, identified ten residues distributed along the pore that
interact with permeant anions and affect anion selectivity, as well as
seven porelining residues that cluster near pore constrictions and
regulate channel gating. Together, these results clarify the basis of
CaCC anion conduction.