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Reconstitution of the influenza virus M2 ion channel in lipid bilayers
M T Tosteson1, L H Pinto, L J Holsinger
1Laboratory for Membrane Transport, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
M2, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M2 baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25-500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25-90 pS). (3) The channels were selective for cations; tNa = 0.75 when 150 mM NaCl bathed both sides of the membrane. (4) Amantadine reduced the probablity of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs approximately Na > Li. The pH activation, amantadine block and ion selectivity of the M2 protein ion channel in bilayers are consistent with those observed on expression of the M2 protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M2 protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.
Insights
The M2 protein from influenza A virus forms ion channels in lipid bilayers. These channels exhibit cation selectivity, pH-dependent activity, and are blocked by amantadine, supporting its role in viral uncoating.
Area of Science:
- Virology
- Membrane Biophysics
- Ion Channel Physiology
Background:
- Influenza A virus M2 protein is crucial for viral replication.
- Its precise function, particularly its ion channel activity, remains under investigation.
Purpose of the Study:
- To characterize the ion channel activity of the purified influenza A virus M2 protein in artificial lipid bilayer membranes.
- To determine the functional properties of the M2 ion channel and compare them to its proposed role in viral uncoating.
Main Methods:
- Purification of M2 protein from infected cells or recombinant baculovirus-infected insect cells.
- Incorporation of purified M2 protein into phospholipid bilayer membranes.
- Electrophysiological recordings to measure ion channel activity, conductance, selectivity, and block.
Main Results:
- Purified M2 protein formed cation-selective ion channels in lipid bilayers.
- Channels displayed burst kinetics with varying conductances, with a predominant low conductance open state.
- Activity was modulated by pH and blocked by amantadine, consistent with in vivo observations.
Conclusions:
- The M2 protein possesses intrinsic ion channel activity.
- These findings support the hypothesis that the M2 protein functions as an ion channel within the influenza virus particle, facilitating viral uncoating.