Abstract
The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform.
Highlights
The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels
The protein was incorporated into small (~100 nm diameter) fusogenic proteoliposomes that subsequently were encapsulated in the aqueous droplet of the DHBs
Reconstitution of MscL-G22E resulted in spontaneous current traces representative of MscL gating (90 pA at +30 mV and 200 mM KCl)[21], verifying that we could reconstitute MscL into DHBs (Fig. 1d)
Summary
The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. This mutant was used to develop a method for controllably activating the channel by injecting buffer into the droplet, thereby stretching the inner DHB monolayer.
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