Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
J Biol Chem. 2019 Aug 16;294(33):12556-12564. doi: 10.1074/jbc.RA118.007128. Epub 2019 Jul 2.
Transmembrane member 16A (TMEM16A) is a widely expressed Ca-activated Cl channel with various physiological functions ranging from mucosal secretion to regulating smooth muscle contraction. Understanding how TMEM16A controls these physiological processes and how its dysregulation may cause disease requires a detailed understanding of how cellular processes and second messengers alter TMEM16A channel gating. Here we assessed the regulation of TMEM16A gating by recording Ca-evoked Cl currents conducted by endogenous TMEM16A channels expressed in oocytes, using the inside-out configuration of the patch clamp technique. During continuous application of Ca, we found that TMEM16A-conducted currents decay shortly after patch excision. Such current rundown is common among channels regulated by phosphatidylinositol 4,5-bisphosphate (PIP). Thus, we sought to investigate a possible role of PIP in TMEM16A gating. Consistently, synthetic PIP rescued the current after rundown, and the application of PIP modulating agents altered the speed kinetics of TMEM16A current rundown. First, two PIP sequestering agents, neomycin and anti-PIP, applied to the intracellular surface of excised patches sped up TMEM16A current rundown to nearly twice as fast. Conversely, rephosphorylation of phosphatidylinositol (PI) derivatives into PIP using Mg-ATP or inhibiting dephosphorylation of PIP using β-glycerophosphate slowed rundown by nearly 3-fold. Our results reveal that TMEM16A regulation is more complicated than it initially appeared; not only is Ca necessary to signal TMEM16a opening, but PIP is also required. These findings improve our understanding of how the dysregulation of these pathways may lead to disease and suggest that targeting these pathways could have utility for potential therapies.
跨膜成员 16A（TMEM16A）是一种广泛表达的 Ca 激活的 Cl 通道，具有多种生理功能，从粘膜分泌到调节平滑肌收缩不等。了解 TMEM16A 如何控制这些生理过程以及其失调如何导致疾病，需要详细了解细胞过程和第二信使如何改变 TMEM16A 通道门控。在这里，我们通过使用膜片钳技术的内向外配置，在卵母细胞中记录内源性 TMEM16A 通道介导的 Ca 诱发的 Cl 电流，评估了 TMEM16A 门控的调节。在连续应用 Ca 的过程中，我们发现 TMEM16A 介导的电流在膜片钳切除后不久就会衰减。这种电流衰减在受磷脂酰肌醇 4,5-二磷酸（PIP）调节的通道中很常见。因此，我们试图研究 PIP 在 TMEM16A 门控中的可能作用。一致地，合成的 PIP 挽救了电流衰减后的电流，并且 PIP 调节剂的应用改变了 TMEM16A 电流衰减的速度动力学。首先，两种 PIP 隔离剂，新霉素和抗 PIP，应用于切除的膜片钳的胞内表面，使 TMEM16A 电流衰减的速度加快了近两倍。相反，使用 Mg-ATP 将磷脂酰肌醇（PI）衍生物重新磷酸化为 PIP 或使用 β-甘油磷酸抑制 PIP 的去磷酸化，使衰减速度减慢了近 3 倍。我们的结果表明，TMEM16A 的调节比最初看起来要复杂；不仅 Ca 是信号 TMEM16a 开放所必需的，而且 PIP 也是必需的。这些发现提高了我们对这些途径失调如何导致疾病的理解，并表明针对这些途径可能对潜在的治疗具有实用价值。
