Department of Chemistry "Ugo Schiff", University of Florence, 50019 Sesto Fiorentino, Italy.
Molecules. 2020 Sep 11;25(18):4167. doi: 10.3390/molecules25184167.
P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca-ATPase, mammalian Cu-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.
P 型 ATP 酶是一类广泛存在于各种生物中的膜转运蛋白，这些酶通过 ATP 水解将各种离子或磷脂跨膜运输，从而产生并维持关键的电化学势梯度。人们已经采用多种方法对 P 型 ATP 酶进行了研究，这些方法提供了大量有关该酶类结构、功能和调控的信息。在用于研究 P 型 ATP 酶的众多技术中，基于固体支撑膜（SSM）的电学法被用于研究各种离子泵的转运机制。特别是，SSM 方法允许在膜结合酶吸附到 SSM 表面并通过底物浓度跃变进行化学激活后，直接测量由 ATP 酶产生的电荷移动。这种测量方法有助于确定生电部分反应并确定膜转运蛋白反应循环中的离子易位位置。在本综述中，我们讨论了 SSM 方法如何有助于研究 P 型 ATP 酶转运机制的一些关键特征，特别关注肌浆网 Ca-ATP 酶、哺乳动物 Cu-ATP 酶（ATP7A 和 ATP7B）和磷脂翻转酶 ATP8A2。
