PDF(Pubmed)
Abstract：
The bacterial mechanosensitive channel of large conductance MscL is activated exclusively by increased tension in the membrane bilayer. Despite many proposed models for MscL opening, its precise mechano-gating mechanism, particularly how the received force at the tension sensor transmits to the gate remains incomplete. Previous studies have shown that along with amphipathic N-terminus located near the cytoplasmic surface of the membrane, Phe78 residue near the outer surface also acts as a \"tension sensor,\" while Gly22 is a central constituent of the \"hydrophobic gate.\" Present study focused on elucidating the force transmission mechanism from the sensor Phe78 in the outer transmembrane helix (TM2) to the gate in the inner transmembrane helix (TM1) of MscL by applying the patch clamp and molecular dynamics (MD) simulations to the wild type MscL channel and its single mutants at the sensor (F78N), the gate (G22N) and their combination (G22N/F78N) double mutant. F78N MscL resulted in a severe loss-of-function, while G22N MscL caused a gain-of-function channel exhibiting spontaneous openings at the resting membrane tension. We initially speculated that the spontaneous opening in G22N mutant might occur without tension acting on Phe78 residue. To test this hypothesis, we examined the (G22N/F78N) double mutant, which unexpectedly exhibited neither spontaneous activity nor activity by a relatively high membrane tension. To understand the underlying mechanism, we conducted MD simulations and analyzed the force transduction pathway. Results showed that the mutation at the tension sensor (F78N) in TM2 caused decreased interaction of this residue not only with lipids, but also with a group of amino acids (Ile32-Leu36-Ile40) in the neighboring TM1 helix, which resulted in an inefficient force transmission to the gate-constituting amino acids on TM1. This change also induced a slight tilting of TM1 towards the membrane plane and decreased the size of the channel pore at the gate, which seems to be the major mechanism for the inhibition of spontaneous opening of the double mutant channel. More importantly, the newly identified interaction between the TM2 (Phe78) and adjacent TM1 (Ile32-Leu36-Ile40) helices seems to be an essential force transmitting mechanism for the stretch-dependent activation of MscL given that substitution of any one of these four amino acids with Asn resulted in severe loss-of-function MscL as reported in our previous work.
摘要：
大电导MscL的细菌机械敏感通道仅通过膜双层中增加的张力而激活。尽管提出了许多MscL开放的模型，其精确的机械门控机构，特别是在张力传感器处接收到的力如何传递到闸门仍然是不完整的。以前的研究表明，随着两亲性N端位于膜的细胞质表面附近，外表面附近的Phe78残留物也用作张力传感器，而Gly22是疏水门的中心组成部分。“本研究的重点是通过将膜片钳和分子动力学（MD）模拟应用于MscL的外跨膜螺旋（TM2）中的传感器Phe78到MscL的内跨膜螺旋（TM1）中的门的力传递机制。野生型MscL通道及其单个突变体在传感器（F78N）上，门(G22N)及其组合(G22N/F78N)双突变体。F78NMscL导致严重的功能丧失，而G22NMscL导致功能增益通道在静息膜张力下表现出自发开口。我们最初推测，G22N突变体的自发开放可能在没有张力作用于Phe78残基的情况下发生。为了检验这个假设，我们检测了(G22N/F78N)双突变体，出乎意料地既没有表现出自发活动，也没有表现出相对较高的膜张力的活动。要了解潜在的机制，我们进行了MD模拟并分析了力传递途径。结果表明，TM2中张力传感器（F78N）的突变不仅导致该残基与脂质的相互作用减少，但在邻近的TM1螺旋中也有一组氨基酸（Ile32-Leu36-Ile40），这导致向TM1上的栅极构成氨基酸的力传递效率低下。这种变化也引起了TM1向膜平面的轻微倾斜，并减小了门处通道孔的大小。这似乎是抑制双突变通道自发开放的主要机制。更重要的是,新发现的TM2（Phe78）和相邻TM1（Ile32-Leu36-Ile40）螺旋之间的相互作用似乎是MscL拉伸依赖性激活的重要力传递机制，因为这四种氨基酸中的任何一种被Asn取代会导致严重的MscL功能丧失。
