Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels

Abstract

TMEM16A and TMEM16B encode for Ca2+-activated Cl− channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B.

Known structures and unknown mechanisms of TMEM16 scramblases and channels

“…The fundamental properties of the TMEM16A and B channels are well characterized: their opening is synergistically driven by depolarizing voltages and binding of Ca 2+ to two conserved sites located within the transmembrane region (Yu et al, 2012; Terashima et al, 2013; Brunner et al, 2014; Tien et al, 2014; Jeng et al, 2016; Lim et al, 2016), their anion selectivity sequence is I − >Br − >Cl − >F − (Caputo et al, 2008; Schroeder et al, 2008; Yang et al, 2008; Pifferi et al, 2009), and they are inhibited by nonspecific Cl − channel blockers such as niflumic acid, 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid, and N -phenylanthranilic acid (Yang et al, 2008; Yu et al, 2012; Scudieri et al, 2015). Prolonged exposures to Ca 2+ induce rundown or desensitization in both channels (Pifferi et al, 2009; Stephan et al, 2009; Ni et al, 2014; Dang et al, 2017; Pifferi, 2017). The mechanisms underlying this process are not clear at the moment, although it has been recently proposed that it might depend on PIP2 depletion (Tembo and Carlson, 2018).…”

Bioinformatic characterization of the Anoctamin Superfamily of Ca2+-activated ion channels and lipid scramblases

“…Yang et al presented evidence that a K584Q mutation in TMEM16A/Ano1 (residue 559 in TMEM16F), alters the anion/cation selectivity [ 43 ], but this result could not be reproduced in a subsequent study [ 65 ]. Although the reasons for this discrepancy are unclear, the evidence available suggests that residues facing the channel pore control both ion selectivity and gating of the channel [ 66 ].…”

Polymodal Control of TMEM16x Channels and Scramblases

“…Anion permeation in TMEM16A requires Cl − binding to a series of positively charged residues within the pore, including K584, R617, and K641 (in aTMEM16A) [108,117,118,122,146]. Mutations of pore residues R573 and K540 in TMEM16B (corresponding to R617 and K584 in aTMEM16A, respectively) lead to alterations in the ion selectivity and Ca 2+ sensitivity of the TMEM16B channel, emphasising that TMEM16A and B channels share similar pore structures and permeation mechanisms [18,21].…”

“…The TMEM16A and B channels have the highest (~60%) sequence homology within the family [12,[14][15][16][17], and present similar electrophysiological properties, including comparable degrees of selectivity and permeability to a range of anions, and sensitivity to intracellular Ca 2+ [1,[18][19][20][21][22][23]. TMEM16A and B channels also share some pharmacological properties; for example, they are modulated in a complex manner by antracene-9-carboxilic acid (A9C) [24,25], and are inhibited by other commonly used Cl − channel blockers-such as 4,4diisothiocyano-2,2 -stilbenedisulfonic acid (DIDS) and niflumic acid (NFA)-to a similar extent [22,26,27].…”