Department of Biology, University of Maryland, College Park, Maryland; Maryland Biophysics Program, University of Maryland, College Park, Maryland.
Department of Biology, University of Maryland, College Park, Maryland.
Biophys J. 2024 Jan 16;123(2):195-209. doi: 10.1016/j.bpj.2023.12.007. Epub 2023 Dec 14.
Mechanosensitive channel MscS, the major bacterial osmolyte release valve, shows a characteristic adaptive behavior. With a sharp onset of activating tension the channel population readily opens, but under prolonged action of moderate tension it inactivates. The inactivated state is non-conductive and tension insensitive, which suggests that the gate becomes uncoupled from the lipid-facing domains. Because the distinct opening and inactivation transitions are both driven from the closed state by tension transmitted through the lipid bilayer, here we explore how mutations of two conserved positively charged lipid anchors, R46 and R74, affect 1) the rates of opening and inactivation and 2) the voltage dependences of these transitions. Previously estimated kinetic rates for opening-closing transitions in wild-type MscS at low voltages were 3-6 orders of magnitude higher than the rates for inactivation and recovery. Here we show that MscS activation exhibits a shallow nearly symmetric dependence on voltage, whereas inactivation is substantially augmented and recovery is slowed down by depolarization. Conversely, hyperpolarization impedes inactivation and speeds up recovery. Mutations of R46 and R74 anchoring the lipid-facing helices to the inner interface to an aromatic residue (W) do not substantially change the activation energy and closing rates, but instead change the kinetics of both inactivation and recovery and essentially eliminate their voltage dependence. Uncharged polar substitutions (S or Q) for these anchors produce functional channels but increase the inactivation and reduce the recovery rates. The data clearly delineate the activation-closing and the inactivation-recovery pathways and strongly suggest that only the latter involves extensive rearrangements of the protein-lipid boundary associated with the uncoupling of the lipid-facing helices from the gate. The discovery that hyperpolarization robustly assists MscS recovery suggests that membrane potential is one of the factors that regulates osmolyte release valves by putting them either on "ready" or "standby" based on the cell's metabolic state.
机械敏感通道 MscS 是细菌主要的渗透物释放阀，表现出一种特征性的自适应行为。通道群体在受到激活张力的急剧作用时很容易打开，但在适度张力的持续作用下会失活。失活状态是无导电性且对张力不敏感的，这表明门与面向脂质的结构域分离。由于明显的开放和失活转变都是由通过脂质双层传递的张力从关闭状态驱动的，因此我们在这里探讨了两个保守的带正电荷的脂质锚点（R46 和 R74）的突变如何影响 1）开放和失活的速率，以及 2）这些转变的电压依赖性。以前在低电压下对野生型 MscS 的开-关转变的估计动力学速率比失活和恢复的速率高 3-6 个数量级。我们发现 MscS 的激活表现出对电压的浅近对称依赖性，而失活则大大增强，恢复则因去极化而减慢。相反，超极化会阻碍失活并加速恢复。将面向脂质的螺旋锚定到内界面的芳香族残基（W）的 R46 和 R74 突变不会显著改变激活能和关闭速率，但会改变失活和恢复的动力学，并基本上消除其电压依赖性。这些锚点的不带电极性取代（S 或 Q）会产生功能性通道，但会增加失活并降低恢复速率。这些数据清楚地区分了激活-关闭和失活-恢复途径，并强烈表明只有后者涉及到与面向脂质的螺旋与门分离相关的蛋白质-脂质边界的广泛重排。超极化有力地促进了 MscS 的恢复这一发现表明，膜电位是通过根据细胞的代谢状态将渗透压释放阀置于“就绪”或“备用”状态来调节渗透压释放阀的因素之一。
