Bacterial survival is predicated on their ability to withstand sudden changes in external osmolarity, and hence most prokaryotic microbes possess mechanosensitive (MS) ion channels that open and release the pressure in response to hyper-osmotic conditions1. Recent outbursts of antibiotic resistance have made it important to understand MS ion channels as potential new antibiotic targets1. One of the best-studied bacterial channels is the large conductance MS channel (MscL), which spans the bacterial plasma membrane. A prominent hypothesis is that membrane curvature may be responsible for opening MscL since the addition of lysophosphatidylcholine (LPC) lipids, which are believed to curve the membrane by inserting in one of the bilayer leaflets, promotes channel activation2 . However, the degree of membrane curvature and the structural rearrangements governing MscL activation are still unknown2.
The aim of this project is to directly quantify and correlate LPC-induced membrane curvature to MscL activity by using simultaneous bilayer fluorescence and single channel current measurements. The technology for performing such measurements, the droplet-hydrogel bilayer3, consists of a supported lipid bilayer formed between an agarose surface and a water droplet. Here we present the reconstitution, verified by current measurements, of MscL in droplet-hydrogel bilayers. Bilayer integrity was confirmed by transmitted light microscopy and capacitance measurements, and bilayer fluidity was verified by Fluorescence Recovery After Photobleaching (FRAP) measurements. Using this setup we plan to quantify LPC-induced membrane curvature by recording the perturbed alignment of lipid-tethered fluorophores using polarized total internal reflection fluorescence (TIRF) microscopy4. We anticipate that the simultaneous recordings of polarized bilayer fluorescence and MscL currents will allow us to establish directly the degree of membrane curvature required for MscL activation.