TMEM 16 chloride channels are two-faced H

The Rockefeller University Press $30.00 J. Gen. Physiol. 2016 Vol. 148 No. 5 367–373 https://doi.org/10.1085/jgp.201611686 367 Elucidating the oligomeric structure of ion channels is central to understanding their function (Marsh and Teichmann, 2015). The vast majority of ion channels are formed by multiple subunits organized as an annulus surrounding a solitary, centrally located ion-conducting pore. For example, all the potassium channels, Cys-loop receptors like nicotinic acetylcholine receptors and GABA receptors, glutamate receptors, acid-sensing ion channels and epithelial sodium channels, cyclic nucleotide-gated and transient receptor potential channels, orai channels, ryanodine receptors and IP3 receptors, connexins, and bestrophins are formed by three to six subunits bordering a single aperture. Similarly, voltage-gated Ca and Na channels have a pore surrounded by four subunits that fused into a single polypeptide during evolution in eukaryotes. However, a small fraction of channels are oligomers, with each subunit forming its own pore. The CLC chloride channels (dimers), aquaporins (tetramers), voltage-gated proton channels (dimers), bacterial porins (trimers), and two-pore Ca channels (dimers of a two-repeat fusion protein) fall into this multiple pore/multiple subunit category. Two papers in this issue of The Journal of General Physiology by Lim et al. and Jeng et al. add another protein to this minority, namely the Ca-activated Cl− channel (CaCC) TMEM16A (also known as ANO1). The findings in these papers raise fundamental questions about the structural principles underlying ion channel pores by suggesting that the Cl− ion conduction pathways are located, not in the center of the protein, as we have come to expect, but rather on the surface of the protein in contact with the membrane bilayer. The anoctamin proteins have recently attracted a great deal of attention because the discovery of their founding members, TMEM16A and TMEM16B, ended a decade-long search for CaCC genes (Pedemonte and Galietta, 2014; Whitlock and Hartzell, 2017). CaCCs are activated by increases in cytosolic Ca and are crucial for many cellular functions including epithelial secretion, regulation of smooth muscle tone, gut motility, neuronal excitability, and nociception. Biochemical and fluorescence resonance energy transfer (FRET) studies have demonstrated that TMEM16A exists as a homodimer, with each subunit containing a Ca-binding site that is crucial for channel opening (Fallah et al., 2011; Sheridan et al., 2011). However, answering the fundamental question of whether the TMEM16A channel has a single pore at the dimer interface, or each subunit has its own pore, has been less straightforward than expected. The two papers in this issue take similar attacks on this question. The investigators create concatemers comprised of two copies of TMEM16A covalently connected in a head-to-tail arrangement by a 31–amino acid flexible linker. By introducing a mutation into one subunit that alters channel function in a way that can be measured electrophysiologically, the contribution of each subunit to the ionic current can be assessed. These investigators first study mutations that alter the ability of Ca to open the channel. The amino acids responsible for Ca binding to TMEM16A had previously been identified by mutagenesis (Yu et al., 2012; Brunner et al., 2014; Tien et al., 2014). Ca is stabilized in its binding site by oxygen atoms contributed by the side chains of four acidic residues in transmembrane domains (TMDs) 7 and 8 (E702, E705, E734, and D738) and an additional glutamic acid (E654) in TMD6. It should be noted that the amino acid numbering in this Commentary has been chosen to coincide with the a,c isoform used by Lim et al. (2016) (four should be added to find the equivalent residue in the Jeng et al. [2016] paper). The TMEM16A splice variants used by the two groups differ by the presence or absence of exon 6, which encodes 4 amino acids. This exon affects the Ca sensitivity of the channel quantitatively, but otherwise, the behavior of the two isoforms is very similar (Ferrera et al., 2009; Xiao et al., 2011). Both groups design a concatemer composed of one WT subunit and one subunit containing a mutation of E702 that significantly reduces the Ca sensitivity of the channel. The Cl− current encoded by constructs containing E702 mutations in one subunit activates in a biphasic manner as Ca concentration is increased. The Ca dose–response curves can be fitted with two EC50 values consistent with the independent activation of each subunit (Jeng et al., 2016; Lim et al., 2016). Although these data provide convincing demonstrations that each subunit can be activated independently, TMEM16 chloride channels are two-faced