1% ± 3.0%, n = 6 and 66.0% ± 3.7%, n = 7, respectively) (Figure 4B). The observations support the notion that protons and Gu+ have a single common pathway and that, in the open state of the channel, R3 creates a barrier in a narrow stretch of the pore to Gu+, while permitting passage of protons. The MTSET adduct attached to cysteine at this position functions as
a barrier to both protons and Gu+, but one that is Selleckchem PF 2341066 less effective than the shorter and bulkier native arginine side chain against Gu+. To permit an omega current through the Shaker K+ channel VSDs requires a “double gap” (absence of arginine in two consecutive positions within S4′s repeating sequence of an arginine at every third position) (Gamal El-Din et al., 2010). If this were also the case in Hv1, then substitution of N4 with arginine in the R3S background should prevent conduction of Gu+ by bracketing the R3 position with arginines: the native R2 immediately before and an introduced R4 immediately after R3. However, we found that, in the R3S background, the N4R mutation (i.e., the double mutation R3S-N4R) not only preserved proton conduction but did not prevent conduction Selleckchem Bioactive Compound Library by Gu+ (Figure S4). This finding suggests that
the narrow part of the Hv1 conducting pathway is particularly short. If R3 were positioned in the narrow part of the pore in the activated state, as its homologs are in the VSDs of Shaker (Larsson et al., 1996, Baker et al., 1998 and Gamal El-Din et al., 2010) and Kv1.2 (Long et al., 2007) K+ channels and Na+ channels (Yang et al., 1996 and Sokolov et al., 2005),
it would be predicted to have a counter-charged partner from one of the other transmembrane segments. The partner residue for R3 in Kv1 channels—a conserved glutamate in the outer third of S2 (E283 in Shaker, E226 in Kv1.2) (Tiwari-Woodruff et al., 2000 and Long during et al., 2007)—is also present in Na+ channels (DeCaen et al., 2008) but is missing in Hv1. Instead, Hv1 has a unique aspartate, D112, located in the middle of S1, which is conserved in proton channels (Figure 1B) and has been predicted by homology modeling to face R3 in the activated state (Ramsey et al., 2010). Mutation of D112 to alanine was earlier shown to shift the conductance-voltage relationship (G-V) strongly in the positive direction (Ramsey et al., 2010). We found that mutation of D112 to serine had such a strong effect that a step to +150 mV evoked outward current of <50 pA at pHi = pHo = 6 (Figure 5A). We also found that the G-V of R3S is substantially shifted in the depolarized direction (Figure 5A). Strikingly, combining the two mutations in the double mutant D112S-R3S shifted the G-V in the negative direction to be close to that of WT channels (Figures 5A and 5B). D112S-R3S was found to retain the pH gradient sensing and external Zn2+ sensitivity of the WT channel (Figure S2), and also to conduct Gu+ like R3S and R3C (Figure S3).
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