The influenza virus M2 proton-selective ion channel activity facilitates virus uncoating, a process that occurs in the acidic environment of the endosome. The M2 channel causes acidification of the interior of the virus particle, which results in viral protein-protein dissociation. The M2 protein is a homotetramer that contains in its aqueous pore a histidine residue (His-37) that acts as a selectivity filter and a tryptophan residue (Trp-41) that acts as a channel gate. Substitution of His-37 modifies M2 ion channel properties drastically. However, the results of such experiments are difficult to interpret because substitution of His-37 could cause gross structural changes to the channel pore. We described here experiments in which partial or, in some cases, full rescue of specific M2 ion channel properties of His-37 substitution mutants was achieved by addition of imidazole to the bathing medium. Chemical rescue was demonstrated for three histidine substitution mutant ion channels (M2-H37G, M2-H37S, and M2-H37T) and for two double mutants in which the Trp-41 channel gate was also mutated (H37G/W41Y and H37G/W41A). Currents of the M2-H37G mutant ion channel were inhibited by Cu(II), which has been shown to coordinate with His-37 in the wild-type channel. Chemical rescue was very specific for imidazole. Buffer molecules that were neutral when protonated (4-morpholineethanesulfonic acid and 3-morpholino-2-hydroxypropanesulfonic acid) did not rescue ion channel activity of the M2-H37G mutant ion channel, but 1-methylimidazole did provide partial rescue of function. These results were consistent with a model for proton transport through the pore of the wildtype channel in which the imidazole side chain of His-37 acted as an intermediate proton acceptor/donor group. The M2 protein of influenza A virus permits protons to enter virus particles during virion uncoating in endosomes, and the M2 channel also causes the equilibration of pH between the acidic lumen of the trans-Golgi network and the cytoplasm (1-3). The activity of the M2 ion channel is inhibited by the antiviral drug amantadine (4 -6). 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 (TM) 1 domain and forms the pore of the channel, and a 54-residue cytoplasmic tail (7). Chemical cross-linking studies (8 -10) and statistical analysis of the ion channel activity of mixed oligomers (11) showed the active state of the M2 ion channel protein to exist minimally as a homotetramer.Despite the small size of the active M2 oligomer, several lines of evidence indicate that ion channel activity is intrinsic to the M2 protein. First, ion channel activity has been observed in three different expression systems, Xenopus oocytes (4, 6), mammalian cells (6, 12), and yeast (13,14). Second, M2 channel activity has also been recorded in artificial lipid bilayers from a reconstituted peptide corresponding to the TM domain of the M2 protein (15) and from purified M2 pr...

Background The serotonin type 3 receptor (5-HT 3 R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family. Results Thirteen amino acids in the mouse 5-HT 3AS R that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT 3 R antagonist [ 3 H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT 3 R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT 3AS R. Conclusion Our data supports a role for the putative E-loop region of the 5-HT 3 R in the binding of 5-HT, m CPBG, d -tc and lerisetron. 5-HT and m CPBG interact with Y142, d -tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

Background: The serotonin type 3 receptor (5-HT 3 R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structurefunction relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family.

Background: Lerisetron, a competitive serotonin type 3 receptor (5-HT 3 R) antagonist, contains five functional groups capable of interacting with amino acids in the 5-HT 3 R binding site. Site directed mutagenesis studies of the 5-HT 3A R have revealed several amino acids that are thought to form part of the binding domain of this receptor. The specific functional groups on the ligand that interact with these amino acids are, however, unknown. Using synthetic analogs of lerisetron as molecular probes in combination with site directed mutagenesis, we have identified some of these interactions and have proposed a model of the lerisetron binding site.

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