Abstract
The M(2) ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, V(rev). We have confirmed the high proton selectivity of the channel (1.5-2.0 x 10(6)) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V(rev) with proton equilibrium potential; 2) measurement of pH(in) and V(rev) while Na(+)(out) was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M(2)-expressing cells with cells exposed to a protonophore. Increased currents at low pH(out) are due to true activation and not merely increased [H(+)](out) because increased pH(out) stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virus-infected cell, experiments employing methods 1-3 show that the channel is also capable of conducting NH(4)(+), probably by a different mechanism from H(+).
Highlights
The M2 protein of influenza A virus is thought to function as an ion channel that permits protons to enter virus particles during virion uncoating in endosomes
The mature M2 protein consists of a 23-residue N-terminal extracellular domain, a single internal hydrophobic domain of 19 residues that acts as a transmembrane domain and forms the pore of the channel, and a 54-residue cytoplasmic tail [6]
The reversal voltage of the currents of cells expressing the M2 protein can be measured from continuous current-voltage relationships measured with ramps of membrane voltage because the M2 ion channel is not voltage-activated on the time scale of the ramps of voltage that are practical to use
Summary
MRNA Synthesis—The cDNA to the A/Udorn/72 mRNA was cloned into the BamHI site of pGEM3 such that mRNA sense transcripts could be generated by using the bacteriophage T7 RNA polymerase promoter and T7 RNA polymerase. Electrodes were filled with 3 M KCl, and the oocytes were bathed in either Barth’s solution, which contained, in mM, 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 NaNO3, 0.71 CaCl2, 0.82 MgSO4, 15 HEPES, pH 7.5, osmolality ϳ210 mosmol/kg or a modified solution during the recording. Control cells or cells infected with rSV40-M2 were incubated (37 °C for 1 h) in a solution containing BCECF-AM in 0.25% Me2SO carrier with a final dye concentration of 0.25 g/ml These cells were placed on the stage of an epifluorescence microscope equipped with a ϫ 20, 0.75 NA (Nikon) objective that allowed up to seven CV-1 cells to be imaged in its field at one time, an intensified CCD camera and MagiCal image analysis software (Applied Imaging, Sunderland, UK). The emission at 520 nm as a result of excitation at 490 and 435 nm was measured while the cell was bathed in solutions with pH spanning the range of pH values expected to be encountered during the measurements (pH 4.0, pH 6.7, and pH 9.0), and the resulting ratios (F490/F435) were used to construct a calibration curve (see Equation 1 of Ref. 26)
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