, 2010) to lie within the span of the membrane at positive voltag

, 2010) to lie within the span of the membrane at positive voltage, so that, in principle, either or both could lie in the pore in

the open state. By analogy with the conductance properties of the Shaker K+ channel VSD, however, the arginine at R3 would be incompatible with the conductance of cations, including protons, but the neutral asparagine at N4 may be compatible. We examined both positions. We began by following up on the earlier finding that the large organic cation, guanidinium (Gu+), blocks proton efflux through the Hv1 channel (Tombola et al., 2008), suggesting that RO4929097 Gu+ might enter the internal mouth of the pore from the internal solution, but might be too large to pass through it. We reasoned that mutations that widened the pore might let Gu+ permeate and would thereby identify the residues that line the pore. In symmetric pH 8.0 100 mM Gu+ solutions, the wild-type (WT) channel did not conduct outward current (Figure 2A and see Figure S1

available online). Mutation of N4 to serine (N4S) did not alter this behavior (Figure 2A). In contrast, mutation of R3 to serine (R3S) led to large voltage-dependent outward currents (Figures 2A and 2B), suggesting that this mutation enables Gu+ to permeate the pore. To test whether Gu+ was actually the permeating cation (and not protons), we measured tail currents evoked by a large depolarizing step, at different tail potentials (Figure 2C). In symmetric pH 8, the reversal potential of the GSI-IX tail currents of R3S depended on the balance between internal and external Gu+ concentrations, closely approximating the predicted equilibrium potential for Gu+ (Figures 2C and 2D). This indicates that Gu+ is the main conducting ion in R3S in symmetric pH 8. We tested the role of R3 in selectivity with mutation to 15 other amino acid identities, including nonpolar, polar, and positively charged side chains. We found that all of the mutants (glycine, alanine, cysteine, leucine, methionine, tryptophan, proline, serine, threonine, asparagine, glutamine, tyrosine, aspartate, histidine, and lysine) support voltage-dependent

current in 100 mM Gu+ Isotretinoin (Figure S1). Thus, the native arginine at R3 is uniquely suited to prevent Gu+ conduction at pH 8. Two defining features of intact proton channels are a high sensitivity to external Zn2+ (Ramsey et al., 2006 and Sasaki et al., 2006) as well as gating that depends on the pH gradient across the membrane (Ramsey et al., 2010). We found that both of these features were preserved in the R3S mutant channel. First, we found that proton current through the R3S mutant measured at symmetric pH 6 was efficiently inhibited by external Zn2+. Application of 100 μM external Zn2+ reduced the proton current by 99.9% ± 0.01% (n = 3) (Figure 3A). This degree of inhibition is similar to what has been observed in the WT channel (Ramsey et al., 2006).

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