Center for Neuroscience, University of California, Davis, California, United States of America.
Department of Neurology, University of California, Davis, California, United States of America.
PLoS One. 2020 Apr 17;15(4):e0231812. doi: 10.1371/journal.pone.0231812. eCollection 2020.
TMEM16A, a Ca2+-sensitive Cl- channel, plays key roles in many physiological functions related to Cl- transport across lipid membranes. Activation of this channel is mediated via binding intracellular Ca2+ to the channel with a relatively high apparent affinity, roughly in the sub-μM to low μM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co2+, which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca2+-induced TMEM16A current by competing with Ca2+ for the aforementioned high-affinity activation sites. In addition, Co2+ also potentiates the Ca2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co2+, similar to our previous findings that high concentrations (mM) of intracellular Ca2+ ([Ca2+]i) can induce more TMEM16A current after the Ca2+-activation sites are saturated by tens of μM [Ca2+]i. The degrees of potentiation by Co2+ and Ca2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co2+ potentiation and the Ca2+ potentiation share a similar mechanism by increasing Cl- flux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.
TMEM16A 是一种钙敏氯离子通道，在许多与氯离子跨脂质膜转运相关的生理功能中发挥关键作用。该通道的激活是通过细胞内钙离子与通道结合来介导的，其结合亲和力相对较高，大致在亚微摩尔到低微摩尔浓度范围内。最近获得的 TMEM16 分子的高分辨率结构表明，高亲和力钙激活位点是由几个酸性氨基酸形成的，它们通过带负电荷的侧链羧基来配位结合的钙离子。在这项研究中，我们研究了 TMEM16A 与二价阳离子 Co2+ 的相互作用，Co2+ 本身不能激活 TMEM16A 的电流。然而，当 Co2+ 被应用于细胞内时，它有两个作用。它通过与 Ca2+ 竞争上述高亲和力激活位点来抑制 Ca2+ 诱导的 TMEM16A 电流。此外，Co2+ 还以低亲和力增强 Ca2+ 诱导的电流。这种增强效应需要高浓度（mM）的 Co2+，类似于我们之前的发现，即当 Ca2+ 激活位点被数十微摩尔 [Ca2+]i 饱和后，高浓度（mM）的细胞内 [Ca2+]i 可以诱导更多的 TMEM16A 电流。Co2+ 和 Ca2+ 的增强效应也大致相互关联。有趣的是，突变 TMEM16A 的一个孔残基 Y589 会改变增强的程度，即被取代的残基的侧链越小，二价阳离子诱导的增强越大。我们认为，Co2+ 的增强效应和 Ca2+ 的增强效应通过增加氯离子通过通道孔的流量具有相似的机制，这可能是由于二价阳离子与孔中的磷脂结合后增加了通道的正孔电位。孔残基的较小侧链可能允许更多的磷脂进入孔内，从而增强高浓度二价阳离子引起的电流增强。
