Equilibrium parameters of spermine block in various Kir2.1* dimeric channel constructs. Data from individual patches were fit to a three-site two-barrier model of spermine block (see Materials and methods), comprising two sequentially linked shallow and deep binding sites. Fit parameters describe the effective valence (A and B) and equilibrium constants (C and D) associated with the shallow (zδ1 and K1) and deep (zδ2 and K2) equilibria. Overall, the modification of position 169C or 176 has prominent effects on deep spermine binding (B and D) and very weak effects on shallow binding (A and C). Note the remarkably weak effects of MTSET modification of position 176C.