Mechanism for proton conduction of the M(2) ion channel of influenza A virus.
The M(2) integral membrane protein of influenza A virus forms a proton-selective ion channel. We investigated the mechanism for proton transport of the M(2) protein in Xenopus oocytes using a two-electrode voltage clamp and in CV-1 cells using the whole cell patch clamp technique. Membrane currents were recorded while manipulating the external solution to alter either the total or free proton concentration or the solvent itself. Membrane conductance decreased by approximately 50% when D(2)O replaced H(2)O as the solvent. From this, we conclude that hydrogen ions do not pass through M(2) as hydronium ions, but instead must interact with titratable groups that line the pore of the channel. M(2) currents measured in solutions of low buffer concentration (<15 mM in oocytes and <0.15 mM in CV-1 cells) were smaller than those studied in solutions of high buffer concentration. Furthermore, the reversal voltage measured in low buffer was shifted to a more negative voltage than in high buffer. Also, at a given pH, M(2) current amplitude in 15 mM buffer decreased when pH-pK(a) was increased by changing the buffer pK(a). Collectively, these results demonstrate that M(2) currents can be limited by external buffer capacity. The data presented in this study were also used to estimate the maximum single channel current of the M(2) ion channel, which was calculated to be on the order of 1-10 fA.
Mould
J A
JA
Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Li
H C
HC
Dudlak
C S
CS
Lear
J D
JD
Pekosz
A
A
Lamb
R A
RA
Pinto
L H
LH
D017738:Alkanesulfonic Acids; D000547:Amantadine / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D017666:Deuterium Oxide; D004553:Electric Conductivity; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D009025:Morpholines; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000378:metabolism*; D014867:Water; D014981:Xenopus
D017738:Alkanesulfonic Acids; D002021:Buffers; D007473:Ion Channels; C031540:M-protein, influenza virus; D009025:Morpholines; D011522:Protons; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D014867:Water; C004550:2-(N-morpholino)ethanesulfonic acid; D000547:Amantadine; D017666:Deuterium Oxide