Novel approach for probing protein-lipid interactions of MscL, a membrane-tension-gated channel

The bacterial mechanosensitive channel MscL, a homo-pentameric protein that resides in the cytoplasmic membrane, protects the cell from acute hypo-osmotic stress by opening in response to increased membrane tension. Early work demonstrated that only the structural protein and a lipid membrane are required for the activity of this channel. Subsequent characterization, much of it directed by the crystallization of the M. tuberculosis protein, focused on a genetic dissection of internal contributions to the channel activity and yielded an intramolecular gating model based on hydrophobic interactions. However, because the membrane matrix provides the medium through which the 'agonist', tension, is delivered to the channel we have inaugurated work to investigate the role of the lipid environment on the activation of this channel. Here we show that the MscL channel activity can be reconstituted in lipid membranes of discrete composition, and that the gating-tension threshold can be determined for these systems. This technique now enables the dissection of the various lipid effects such as lateral pressure, bilayer thickness, and head-group chemistry, on the function of a membrane protein. Here we describe the methodology required to address this question and present preliminary data demonstrating a specific lipid effect on the function of this mechanosensitive channel.