If D112 interacted with R3, as deduced above, then one would predict that a substitution of the aspartate at D112 with glutamate would keep a relatively normal G-V because it retained the negative charge. This was indeed observed in the single mutant D112E (Figure 5A). If our inferences so far are correct, and R3 and D112 interact in the open conformation, with R3 lining the pore, then Selleck Natural Product Library D112 would also be expected to line the

open pore. We tested this expectation by considering our observation that substitution of R3 with any of a variety of different amino acids leads to outward current in the high Gu+ solution, i.e., loss of ion selectivity (Figure S1). This led us to predict that a mutation at R3 that has lost ion selectivity should have the selectivity

restored if a complementary mutation could be made at D112 that would reestablish an interaction. If the interaction between D112 and R3 were electrostatic, then a charge reversal would provide a good test. Since, as seen with the 14 other substitutions made initially at R3, a mutation of R3 to aspartate (R3D) also compromises ion selectivity, giving rise to outward current at 100 mM Gu+ at pH 8 (Figures S1 and 6), we tested the effect of a charge reversal. Strikingly, the addition of the mutation D112R to the selectivity-compromised R3D to generate the double mutant find more D112R-R3D (thus swapping the charges between D112 and R3) yielded a channel with an outward current at pHi = 6, pHo = 8, but not with 100 mM Gu+ pH 8 as the internal solution (Figure 6). In other words, the introduction of the charge reversal by the D112R mutation complemented the effect of the R3D charge reversal until and restored proton selectivity (Gu+ exclusion), as predicted for an interaction between D112 and R3 in the open state of the channel. Based on what we have seen so far, the contribution of D112 to ion selectivity could be explained by an indirect effect of D112 on the role of R3 in selectivity. We next set out to determine if D112 has a direct effect on selectivity. To do this we extended our analysis to metal cations and compared the

conductance of WT channels and channels mutated at either R3 alone or both R3 and D112. Our first approach was to test outward currents elicited by voltage steps (to +60 mV) in patches where the pipette was filled (external solution) with 100 mM NaCl solution and to sequentially test 100 mM internal Na+, Li+, K+, Cs+, and Gu+. The experiment was carried out in symmetric pH 8 to minimize contribution by proton current. WT channels supported outward current in the presence of the metal cations, with modestly larger currents in Na+, K+, and Cs+ than in Li+ (Figure 7A). In Gu+, current was almost entirely abolished (reduction to 8.6 ± 1.4%, n = 8) (Figure 7A), consistent with pore block, as shown above (Figure 2A and S1).