e., Na+ plus protons). We observed little change in Na+ current (Figure S5), suggesting that most of the current of WT channels in the presence of metal ions is carried by protons (Figure 7A, dashed line). In contrast to WT, R3S channels had currents in Gu+ that were almost 9-fold larger than in Li+ (Figure 7B). Moreover, unlike WT, in the presence of metal ions, R3S current was largest in Li+ (Figure 7B) (e.g., the K+/Li+ ratio was 1.32 ± 0.07 for WT versus 0.24 ± 0.02 for R3S, n = 8 and

n = 11, respectively, p < 0.01, t test). The 100 mM TRIS pH 8 (protons alone) versus 100 mM Na+ pH 8 (i.e., Na+ plus protons) ratio was also close to unity in R3S (Figure S5), suggesting that the R3S mutation increases permeability to Li+ (Figure 7B, bottom, dashed line). To examine D112 we first turned to the D112S see more mutant, but its current was too small (Figure 5). Since the charge conserving D112E mutation did not shift the G-V (Figure 5), the substituted glutamate of this mutant seems

to accommodate the normal interactions of the native aspartate. If the model was correct and pairing between R3 and D112 were important for selectivity, one would expect the D112E mutant to retain normal selectivity. This was indeed the case. The D112E mutant had no appreciable conductance in Gu+ and the order of current amplitudes in the different metal cations closely resembled that of WT (Figure 7D). We therefore turned to the D112S-R3S double mutant. In D112S-R3S, the current of Gu+ was more than 14-fold larger than that of Li+ (Figure 7C). This value is significantly larger than what is seen in R3S alone (Gu+ / Li+ ratio: R3S, 8.69 ± 0.45, Selleck PLX4032 n = 11; D112S-R3S, 14.25 ± 1.77, n = 8; p < 0.01, t test). Strikingly, assessment of the protons alone versus Na+ plus protons ratio indicated that, unlike WT and R3S channels, most of the D112S-R3S current in presence of Na+ is

actually carried by Na+ (Figure S5; the proton/[proton + 100 mM Na+] ratio was 0.22 ± 0.03, n = 5 for D112S-R3S, significantly different from both 0.82 ± 0.06, n = 7 for R3S and 0.80 ± 0.05 for WT, p < 0.01, ANOVA followed by Dunn's method Cediranib (AZD2171) for multiple comparison). To test more precisely the effects on ion selectivity of R3 and D112, we examined the reversal potentials of tail currents under mono- and bi-ionic conditions. In WT channels there was no Gu+ conduction and reversal potentials did not differ between Na+ and Li+ (Erev shift = 0.57 ± 1.20 mV, n = 4, p = 0.67, paired t test), consistent with the analysis above, that indicated that in Na+ and Li+ the current is mainly carried by protons. In R3S the reversal potential shift between Na+ and Li+ was larger and statistically significant (Erev shift = −4.24 ± 1.70 mV, n = 8, p = 0.04, paired t test). In D112S-R3S the reversal potential shift between Na+ and Li+ increased even more (Erev shift = −13.91 ± 2.30 mV, n = 5, p < 0.

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