The ability to functionalize droplet interface bilayers (DIBs) with the MscL channel and its mutants has been demonstrated. In previous work, the V23T gain of function mutant of MscL produced consistent activation when harmonic axial compressions were applied to the aqueous droplets supporting the lipid bilayer, where the channels settle. The deformation of the droplets results, at maximum compression, in an increase in surface area, and thus an increase in tension at the water-lipid-oil interface. This increase in monolayer tension was found to be the product of the relative change in surface area of each of the droplets and the compressibility modulus of the DPhPC monolayer (∼120 mN/m). The tension increase at the water-lipid-oil interface almost doubles to make up the increase in tension in the bilayer interface, resulting in activation of the incorporated MscL channels. However, it was found that the application of a relatively high transmembrane potential (∼100 mV), from an external power source, is a requirement for the activation of the V23T-MscL channels. Here, we investigate and analyze the impact of transmembrane potential on the activity of MscL channels in both a droplet interface bilayer system and E. coli spheroplast via patch-clamp. We demonstrate that the channels became more susceptible to gating upon the application of a negative potential, compared to when a positive potential is applied, proving their sensitivity to voltage polarity.