Fig. 5. Effect of representative filter mutations on the S331P GOF mutant. (A) Additional G187Q (S331P-G187Q) mutation on the K⁺ filter rescues the S331P mutant on K200 plates (bottom row). The Y188F (S331P-Y188F) mutation is the only K⁺ filter mutation we could isolate that did not rescue the S331P mutant on K200 plates (row 3). (B) Western blot analysis of the membrane fractions prepared from the cells carrying vectors: pGEM-empty (lane 1), pGEM-WT (lane 2), pGEM-S331P (lane 3) and pGEM-S331-G187Q (lane 4). All strains produce a comparable amount of Kch and RCK proteins in the bacterial membrane. RCK: RCK protein (see Figure 6A). M: MagicMark (Invitrogen, CA).