The M2 protein from the influenza A virus, an acid-activated proton-selective channel, has been the subject of numerous conductance, structural, and computational studies. However, little is known at the atomic level about the heart of the functional mechanism for this tetrameric protein, a His37-Trp41 cluster. We report the structure of the M2 conductance domain (residues 22 to 62) in a lipid bilayer, which displays the defining features of the native protein that have not been attainable from structures solubilized by detergents. We propose that the tetrameric His37-Trp41 cluster guides protons through the channel by forming and breaking hydrogen bonds between adjacent pairs of histidines and through specific interactions of the histidines with the tryptophan gate. This mechanism explains the main observations on M2 proton conductance.

The heart of the H ؉ conductance mechanism in the homotetrameric M2 H ؉ channel from influenza A is a set of four histidine side chains. Here, we show that protonation of the third of these imidazoles coincides with acid activation of this transmembrane channel and that, at physiological pH, the channel is closed by two imidazole-imidazolium dimers, each sharing a low-barrier hydrogen bond. This unique construct succeeds in distributing a pair of charges over four rings and many atoms in a low dielectric environment to minimize charge repulsion. These dimers form with identical pK as of 8.2 ؎ 0.2, suggesting cooperative H ؉ binding and clearly illustrating high H ؉ affinity for this channel. The protonation behavior of the histidine side chains has been characterized by using solid-state NMR spectroscopy on the M2 transmembrane domain in fully hydrated lipid bilayers where the tetrameric backbone structure is known. Furthermore, electrophysiological measurements of multichannel and single-channel experiments confirm that these protein constructs are functional.M2 channel ͉ proton channel ͉ solid-state NMR ͉ low-barrier hydrogen bond ͉ histidine ionization constants A histidine tetrad in the pore of the tetrameric M2 protein has long been associated with key channel features of H ϩ selectivity, pH activation, gating, inhibition, and the specific conductance mechanism. M2 protein from influenza A virus conducts protons into the viral core after endocytosis, which leads to the uncoating and release of genetic material into the cytoplasm after fusion of the viral coat with the endosomal wall (1, 2). Much is known about this system from its tetrameric state (2-4), the backbone structure of the transmembrane (TM) domain (5), and numerous electrophysiological (6, 7), biophysical (8-10), and modeling (11) studies that have cast a fascinating tale for this important influenza drug target and the only proton channel of its kind to be characterized in such detail. However, the specific role of His-37 in the tetrameric protein has not been elucidated. Here, we have characterized the pK a s associated with this cluster of four histidine residues in the hydrophobic interstices of the membrane. These pK a values have led us to substantial mechanistic conclusions.There are many lines of evidence, reviewed by Kelly et al. (6), that support the conclusion that M2 is responsible for viral acidification. In vivo ion conductance recordings have shown pH sensitive conductance resulting in rapid acidification of the Xenopus oocytes (12, 13) and mammalian cells (13-15) containing M2 protein.Preparations of purified M2 protein have also been used to show proton conductance in synthetic lipid bilayers (16,17). Singlechannel conductance measurements with membranes containing M2 protein give clear evidence that it is H ϩ conductance, not counterion conductance, that is observed. Furthermore, the channel conductance is unchanged by addition of an excess of NaCl (18). Conductance measurements for the isolated TM domain of M2 protein have als...

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.

Dipoles of the tryptophan indole side chains have a direct impact on ion conductance in the gramicidin channel. Here, fluorination of the indoles (both 5- and 6-fluoro) is used to manipulate both the orientations and the magnitudes of the dipoles. The orientations and positions with respect to the channel axis were determined using (2)H solid state NMR of uniformly aligned lipid bilayer preparations. By exchange of the remaining four protons in the indole ring for deuterium, comparison could be made to d(5)-indole spectra that have previously been recorded for each of the four indoles of gramicidin A. After making the assignments which were aided by the observation of (19)F-(2)H dipolar interactions, we found that fluorination caused only minor changes in side chain conformation. With the high-resolution structural characterization of the fluorinated indoles in position 11, 13, and 15, the electrostatic interactions with a cation at the channel and bilayer center can be predicted and the influence of the modified dipoles on ion conductance estimated. The importance of the long-range electrostatic interaction was recently documented with the observation of alpha-helical dipoles oriented toward the bilayer center on the ion conductance pathway for the Streptomyces K(+) channel. We present direct measurements of the orientation of gramicidin channel F-Trp positions for use in analysis of dipole effects on channel permeation.