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Previously, it was shown that expression of potassium channels in potassium transport deficient E. coli strains restores growth defect in low potassium media (<5 mM) and allows for isolation of mutations that result in channel activation (1). Using similar principle we isolated potassium leaky mutants of mechanosensitve non-selective channel MscS (2) The mutations that result in potassium leak could be grouped into two major categories: (i) mutations that cause channel to open easier (gain-of-function, GOF) located on the pore-forming helix (TM3); (ii) mutations that result in inactive channels (loss-of-function, LOF) located in peripheral crevices in between TM3 and peripheral helices TM1/2. It raises the question whether potassium leak pathways are the same in GOF and LOF mutants. Here we present analysis of growth temperature dependence of potassium transport deficient E. coli strains expressing GOF and LOF mutants that allows us to identify channel pore as a common pathway for both types of mutants. The results shed also new light on the gating principles of the MscS channel. (1) Sun S, Gan JH, Paynter JJ, Tucker SJ (2006) Physiol Genomics 26: 1–7. (2) Koprowski P, Grajkowski W, Isacoff EY, Kubalski A (2011) J Biol Chem 286: 877–888.