Definitive assignment of proton selectivity and attoampere unitary current to the M2 ion channel protein of influenza A virus

Abstract

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer--1.2 aA (1.2 x 10(-18) A) at neutral pH and 2.7 to 4.1 aA at pH 5.7--which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na+ or K+ probes. Even low-pH-activated M2 did not conduct Na+ and K+. The proton selectivity of M2 was estimated to be at least 3 x 10(6) (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.

FIG. 1

Characterization of isolated and liposome-reconstituted M2 protein. (A) SDS-PAGE (12.5% polyacrylamide) of M2 preparations (I, II, and III) stained with Coomassie blue. Left lane: Amersham RPN800 molecular mass markers (in kilodaltons). (B) Aligned Western blots of the same gel, developed with antiserum to the M2 N terminus (left panel) or C terminus (right panel). 1, monomer; 2, dimer; 4, tetramer. (C) Native 1% agarose gel. Lane 1, 500 ng of M2 plus 40 mM OG; lane 2, protein standard HDL-LDL; lane 3, 250 ng of M2 plus 0.34% Coomassie blue; lane 4, 250 ng of M2, 0.05% taurodeoxycholate, and 40 mM OG. In the right panel, the gel was stained with a cholesterol detection kit to visualize the protein standard in lane 2 (LDL in the upper band and HDL in the lower band). Lane 3 shows prestained M2 and unbound Coomassie blue at the front indicated by a line. The left panel shows an aligned Western blot, developed with anti-M2 rabbit serum. <, loading pockets. (The HDL-LDL standard contained a nonspecifically reacting band migrating to the cathode.) (D) Orientation of liposome-reconstituted M2. Serial twofold dilutions of M2 vesicles, prepared with NaPS (Na⁺) or KPS (K⁺), digested with trypsin in the absence (+) or presence of 40 mM OG (OG), and untreated controls (−) were dot blotted and developed with antiserum to the M2 N terminus (upper panel) or C terminus (lower panel).

FIG. 2

Experimental setup for analysis of M2 activity and ion selectivity. (A) M2 vesicles containing either potassium or sodium ions and a fluorescent pH indicator are introduced into assay buffers, which impose metal ion or pH gradients. The M2 protein is present in both orientations. Metal ion fluxes coupled to proton counterflow are monitored via internal pH. (B) M2 vesicles containing sodium ions are introduced into a buffer containing potassium ions. If no pH change is observed, an ionophore specific for the internal metal ion is added to elicit proton flux (arrow). Addition of monensin (m) supports escape of Na⁺ ions, enabling proton influx through M2. (C) Addition of an ionophore specific for the external metal ion, valinomyin (v), supports K⁺ ion influx, enabling proton efflux through M2. (D) Introduction of M2 vesicles into a buffer lacking both metal ions.

FIG. 3

Cation selectivity of the M2 ion channel in the presence of Na⁺ and K⁺ ions. (A) M2 (solid symbols) or control vesicles (open symbols) containing NaPS were introduced into Na⁺- or K⁺-containing buffer. (B) M2 (solid symbols) or control vesicles (open symbols) containing KPS were introduced into Na⁺ or K⁺ buffer. Fluorimetrically monitored internal pH is plotted against time; the initial pH is 7.4 on the outside of the membrane. The experimental setup is explained in the legend to Fig. 2C and D. Addition of ionophores is indicated by an arrow. Incubation buffers and added ionophores are displayed in the box. Val, valinomycin; mon, monensin.

FIG. 4

Cation selectivity of the M2 ion channel in the presence of a single metal ion. M2 or control vesicles containing NaPS (A) or KPS (B) were introduced into metal ion-free NMDGH buffer, as shown schematically in Fig. 2D. Monensin (A) or valinomycin (B) was added after 10 s (arrow). ΔpH = pH_in − pH_in (t = 0 s). The initial pH is 7.4 on both sides of the membrane. Other symbols are as introduced in the legend to Fig. 3.

FIG. 5

Effect of acidic pH on cation selectivity of the M2 ion channel protein. Vesicles prepared in NaPS (pH 7.4) were introduced into NaPS or KPS at pH 5.7. The data are presented as plots of differences between recordings on M2 vesicles and control (c) vesicles: ΔpH = pH_in(M2) − pH_in(c). Ionophores were added at 20 s (arrow). Incubation conditions: ▴, KPS, pH 5.7 (plus valinomycin); ■, NaPS, pH 5.7 (plus monensin); ●, KPS, pH 5.7 (plus monensin).

FIG. 6

Inhibition of the M2 proton channel activity by rimantadine. Vesicles prepared in KPS (pH 7.4) were introduced into NaPS (pH 7.4), with 1 μM rimantadine, at 18°C and incubated for 5 min (●) before addition of valinomycin to initiate proton translocation. Inhibition without preincubation (○) was observed by introducing vesicles into NaPS containing valinomycin and rimantadine. The uninhibited reaction (▴) was recorded in NaPS (pH 7.4), and the background (▵) was recorded in KPS (pH 7.4).

FIG. 7

Temperature dependence of the proton translocation rate. (A) Proton translocation into M2 and control (c) vesicles. ΔpH = pH_in (t = 0) − pH_in (t = 1 s). (B) Arrhenius plot. The initial proton translocation rate (v [M s⁻¹]) was calculated from ΔpH_in as described in Table 1; log v was plotted against 1/T.