Removal of external fixed negative charges reduces affinity for Mg²⁺ block and K⁺ permeation. (A) Homology model of the outer mouth of Kir1.1b showing the positions of four negatively charged amino acid side chains, E92, D97, E104, and E132, relative to the conduction pathway. (B) Analysis of block by 3 mM Mg²⁺ of wild-type (WT) ROMK and the mutant E104S. The internal solution contained 110 mM K⁺. The external solution contained 11 mM K⁺ plus 99 mM Na⁺. Lines represent fits to Eqs. 2 and 3, with K_Mg(0) = 3.9 mM and zδ_Mg = 0.16 (WT), and K_Mg(0) = 20 mM and zδ_Mg = 0.16 (E104S). (C) Analysis of block by 3 mM Mg²⁺ of WT ROMK and the quadruple mutant E92A/ D97V/ E104S/E132Q. The internal solution contained 110 mM K⁺. The external solution contained 11 mM K⁺ and no Na⁺. Lines represent fits to Eqs. 2 and 3, with K_Mg(0) = 5.2 mM and zδ_Mg = 0.20 (WT), and K_Mg(0) = 9.3 mM and zδ_Mg = 0.17 (E92A/ D97V/ E104S/E132Q). (D) Current traces for WT Kir1.1b in excised inside-out patches with different K⁺ concentrations on both sides of the membrane and 0.5 mM EDTA in the pipette solution. (E) Conductance as a function of concentration for WT and E92A/ D97V/ E104S/E132Q in the presence and absence of 0.5 mM EDTA. Lines represent best fits to Eq. 1, with g_max = 50 pS ± 1 and K_m = 11.0 ± 1.3 mM (WT); g_max = 53 ± 1 pS and K_m = 2.2 ± 0.4 mM (WT + EDTA); g_max = 47 ± 1 pS and K_m = 12 ± 1 mM (E92A/ D97V/ E104S/E132Q); and g_max = 47 ± 2 pS and K_m = 12 ± 2 mM (E92A/ D97V/ E104S/E132Q + EDTA).