Fig. 3. Membrane asymmetry gates MscL G22C F93W channels reconstituted into DIBs. (A) Asymmetric MscL DIBs containing 15 mol% LPC are formed by incubating LPC-containing vesicles in the left droplet of the DIB (PC : PG : LPC 80 : 5 : 15) and MscL-containing vesicles in the right droplet (PC : PG : LPC 95 : 5 : 0). Each 1 μl droplet additionally contained 20 mM HEPES, 100 mM KCl pH 7.4 buffer. The activation of sub conducting states was observed, clustered into three main gating events ∼14, 21 and 42 pA that are attributed to sub-conducting states of the MscL channel (n = 4). Further events for 15 mol% LPC discussed in Fig. 4. (B) Reducing the LPC concentration in the asymmetric DIB attenuates channel function. An ∼10-fold drop in gating events are observed when LPC is reduced to 10 mol%, and no fully open states are recorded for the MscL channel (n = 3). (C) If a symmetric MscL DIB is generated without LPC, no gating events are observed indicating that the MscL channel is closed (n = 2). (D) If asymmetric LPC DIBs are prepared without MscL, no gating events are observed, indicating that in (A) flux across the bilayer is controlled by the response of MscL to membrane asymmetry (n = 3). Cartoons illustrate lipid and protein compositions. Black traces show examples of full, 50 kHz recorded trace for each composition with featured zoom in to indicate channel activity. Zoom in data shown with an applied low pass filter at 2.5 kHz. All traces recorded at −100 mV. The percentage standard error of the mean is denoted by % SE.