Cui C, Smith D O, Adler J
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
J Membr Biol. 1995 Mar;144(1):31-42. doi: 10.1007/BF00238414.
Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 +/- 25 pS and 350 +/- 14 pS in 400 mM symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca2+ and glutamate-. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mechanosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.
对大肠杆菌的巨大原生质体进行了全细胞膜片钳记录。研究了原生质体的压力敏感性。在向细胞内部施加正压或对细胞进行渗透压休克时，在400 mM对称KCl溶液中观察到两种不同单位电导的机械敏感通道，分别为1100±25 pS和350±14 pS。1100 pS电导通道对所测试的单价离子区分能力较差，并且对Ca2+和谷氨酸-具有通透性。这两种通道都对跨膜渗透压梯度敏感；随着渗透压梯度增加，细胞平均电流增加，而通道的单位电导保持恒定。两种通道都对电压敏感。电压斜坡结果表明，原生质体的压力敏感性取决于电压：去极化时比超极化时有更多的通道激活。机械敏感通道可被钆离子可逆性阻断。它们也可被质子可逆性抑制。钾离子外流系统KefB和KefC中的两个突变不影响通道活性，而KefA基因的无效突变则显著改变通道活性。这表明KefA对这些通道具有潜在的调节作用。
