Following the Global Structural Changes of an Ion Channel During its Gating by using a Novel Mass Spectrometry Approach

Abstract

Mechanosensitive ion channels are pore forming membrane proteins playing vital roles in all forms of life. They sense the mechanical force in the lipid bilayer and translate this force into big structural changes. Revealing these structural changes, thus; how these channels work is of great importance for understanding mechanosensation.Mechanosensitive channel of large conductance (MscL) is such a channel in bacteria, which opens a temporary pore as large as 3-4 nm in diameter in response to hypoosmotic shock. In order to form such a big opening, the channel undergoes drastic structural rearrangements. The methods currently used to study MscL gating such as patch clamp, disulfide crosslinking, FRET spectroscopy, SDSL-EPR enabled researchers to gain information on the local structural changes taking place during channel gating. However, a method for direct observation of the overall global structural changes is lacking. Here, we developed a novel approach to track the global structural changes taking place when MscL goes from the closed to the open state. Our method is based on determining the mass and rotationally averaged size of the ion channel in its intact form using non-denaturing electrospray ionization coupled with ion mobility mass spectrometry (IM-MS). We studied native MscL in its closed form and heteropentameric MscLs in different open states. We could detect for the first time i) the native mass, hence the oligomeric state, of MscL; ii) the global structural changes during MscL gating; and iii) functioning of MscL in the absence of a lipid bilayer. We believe our novel approach opens new avenues for further studies on the dynamic structures of many other membrane proteins, which were so far unattainable by other methods.