Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel

Abstract

The A/M2 protein of influenza A virus forms a tetrameric proton-selective pH-gated ion channel. The H(37)xxxW(41) motif located in the channel pore is responsible for its gating and proton selectivity. Channel activation most likely involves protonation of the H37 residues, while the conductive state of the channel is characterized by two or three charged His residues in a tetrad. A/M2 channel activity is inhibited by the antiviral drug amantadine. Although a large number of functional amantadine-resistant mutants of A/M2 have been observed in vitro, only a few are observed in highly transmissible viruses in the presence or absence of amantadine. We therefore examined 49 point mutants of the pore-lining residues, representing both natural and nonnatural variants. Their ion selectivity, amantadine sensitivity, specific activity, and pH-dependent conductance were measured in Xenopus oocytes. These measurements showed how variations in the sequence lead to variations in the proton conduction. The results are consistent with a multistep mechanism that allows the protein to fine-tune its pH-rate profile over a wide range of proton concentrations, hypothesized to arise from different protonation states of the H37 tetrad. Mutations that give native-like conductance at low pH as well as minimal leakage current at pH 7.0 were surprisingly rare. Moreover, the results are consistent with a location of the amantadine-binding site inside the channel pore. These findings have helped to define the set of functionally fit mutants that should be targeted when considering the design of novel drugs that inhibit amantadine-resistant strains of influenza A virus.

Figure 1

A representative trace of the current-voltage relationship (I/V) of wt A/M2 channel activity measured in Xenopus oocytes. A/M2 channels were activated by the application of the acidic bathing medium (pH 5.5) for 10 sec and the channel activity was assayed applying the voltage ramp from −60 to +80 mV to obtain an I/V curve (see Materials and Methods for details). The channel activity was assayed first in the Na⁺ based medium (first blue trace). After 3 min recovery at pH 8.5 the channel activity was assayed again in the NMDG⁺ based medium (red trace) and finally the an additional trace of activity was measured in the Na⁺ based medium after 3 more min recovery (second blue trace) to ensure that the channel properties and the experimental conditions did not change during the experiment. The I/V curves were plotted after the subtraction of the endogenous oocyte currents recorded at pH 8.5. The Vrev of the A/M2 channels measured in Na⁺, or in NMDG⁺ based medium were found to be indistinguishable.

Figure 2

Summary of the relative specific activities of the A/M2 mutants with mutations of pore-lining residues. For generating the plots the amount of channel protein expressed on the oocyte membrane was assayed by immunofluorescence (see Materials and Methods for details) and the whole-cell conductance for each oocyte measured at pH 5.5 was expressed as a function of its protein expression level. The bars represent the slope of the fitted curve, normalized to that of the wt channel. Each bar represents data from 15-20 oocytes and is the mean (± SD) of 3 independent experiments. NF – non functional channel. NS – non selective channel.

Figure 3

Summary of the pH-dependent proton conductance of A/M2 channels with mutated V27, A30, or S31 residues. Left: channel activity of the pore-lining residue mutants (V27, A; A30, B and S31, C) was measured in Xenopus oocytes in the pH range from 5.0 to 8.0. The channel activity was normalized to the specific activity of each mutant measured at pH 5.5 (see results and Figure 2). The data for were fitted using Eq. 2. Each point is the mean (±SD) of 10-15 oocytes from 3 independent experiments. Right: tables representing the pK₁, pK₂, r₁, r₂ values (±SD) obtained from the fits on the left using Eq. 2 (see Results).

Figure 4

Summary of the pH-dependent proton conductance of A/M2 channels with mutated G34, W41, or D44 residues. Left: channel activity of the pore-lining residues mutants (G34, A; W41, B and D44, C) was measured in Xenopus oocytes in the pH range from 5.0 to 8.0. The plots were generates as described in the legend of Figure 3. Each point it a mean (±SD) of 10-15 oocytes from 3 independent experiments. Right: tables representing the pK₁, pK₂, r₁, r₂ values (±SD) obtained from the fits on the left using Eq. 2 (see Results).

Figure 5

Root mean square deviations (RMSDs) of the heavy atoms of M2TM in the MD simulations of the wild type protein and the mutants S31N, V27K, A30K, A30F and S31F. Shown are the RMSDs of the whole protein (red) and of the histidine cluster (blue). Snapshots from the dynamics are superimposed to the RMSD plots with grey, cyan, and orange denoting the protein backbone, the mutated side chains, and the His37 side chains respectively. The angle between the N_ε-N_δ vector and the membrane normal for the His37 side chains (averaged over all four histidines), is also plotted as a function of time (green). In the wild-type and in the S31N and V27K mutants the histidines remain ordered (RMSD around 1 Å), with the N_ε-N_δ vector mostly perpendicular to the membrane plane (the angle is stable around 155°). However, the non-selective A30K mutant and the non-conducting mutants A30F and S31F feature a distorted histidine cluster (RMSD approaching 2 Å or more) and a reduction in the angle with the membrane normal to 120° or less.

Figure 6

The density of water oxygen in the pore as a function of transmembrane displacement (z) is shown along with the structure of the bundle and of selected pore-lining residues (only three chains shown for clarity). A. WT (red), S31N (blue), and V27K (orange); B. WT (red), A30F (blue) and S31F (orange); A30, F30, H37, and W41 are represented as sticks. V27, K27, S31, N31, H37, and W41 are represented as sticks. The region of vanishing water density in A30F and S31F is expanded for clarity.