Pifferi Simone
Neurobiology Group, SISSA, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy.
PLoS One. 2017 Jan 3;12(1):e0169572. doi: 10.1371/journal.pone.0169572. eCollection 2017.
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.
跨膜蛋白16A（TMEM16A）和跨膜蛋白16B（TMEM16B）编码钙离子激活的氯离子通道（CaCC），在多种细胞类型中表达，并在许多生理过程中发挥重要作用。在此，我进行了一项定点诱变研究，以了解TMEM16B离子通透的分子机制。我分别对位于假定通道孔入口处和内部的两个带正电荷的残基R573和K540进行了突变，并使用全细胞和内面向外膜片钳实验测量了在人胚肾293（HEK-293）细胞中表达的野生型和突变型TMEM16B通道的特性。我发现有证据表明，R573和K540控制TMEM16B的离子通透性，这既取决于离子置换发生在膜的哪一侧，也取决于通道激活的水平。此外，这些残基有助于控制通透阴离子的阻断或激活。最后，R573突变消除了在存在可渗透阴离子时观察到的异常摩尔分数效应，并改变了通道的表观钙离子敏感性。这些发现表明，面向假定通道孔的残基既负责控制通道的离子选择性，也负责控制通道的门控，这为初步了解TMEM16B中的离子通透分子机制提供了依据。
