Aromatic residues at position 55 of rat alpha7 nicotinic acetylcholine receptors are critical for maintaining rapid desensitization

The rat α7 nicotinic acetylcholine receptor (nAChR) can undergo rapid onset of desensitization; however, the mechanisms of desensitization are largely unknown. The contribution of a tryptophan (W) residue at position 55 of the rat α7 nAChR subunit, which lies within the β2 strand, was studied by mutating it to other hydrophobic and/or aromatic amino acids, followed by voltage-clamp experiments in Xenopus oocytes. When mutated to alanine, the α7-W55A nAChR desensitized more slowly, and recovered from desensitization more rapidly, than wildtype α7 nAChRs. The contribution of desensitization was validated by kinetic modelling. Mutating W55 to other aromatic residues (phenylalanine or tyrosine) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (alanine, cysteine or valine) significantly decreased the rate of onset and increased the rate of recovery from desensitization. To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of W55 by mutating W55 to cysteine (α7-W55C) and testing the ability of various sulfhydryl reagents to react with this cysteine. Several positively charged sulfhydryl reagents blocked ACh-induced responses for α7-W55C nAChRs, whereas a neutral sulfhydryl reagent potentiated responses; residue C55 was not accessible for modification in the desensitized state. These data suggest that W55 plays an important role in both the onset and recovery from desensitization in the rat α7 nAChR, and that aromatic residues at position 55 are critical for maintaining rapid desensitization. Furthermore, these data suggest that W55 may be a potential target for modulatory agents operating via hydrophobic interactions.     

Role of the DExH motif of the Japanese encephalitis virus and hepatitis C virus NS3 proteins in the ATPase and RNA helicase activities

The role of the conserved DExH motif of the Japanese encephalitis virus (JEV) NS3 protein in the ATPase and RNA helicase activities was compared with that of the hepatitis C virus (HCV) NS3 protein. In the DExH motif of JEV NS3, Asp-285 and Glu-286 were essential for both ATPase and RNA helicase activities. Cys-287 was critical for the RNA helicase activity of JEV NS3 but not for ATPase activity. A His-288-to-Ala substitution in the DExH motif of HCV NS3 resulted in an increase in ATPase activity which was suppressed by poly(U). In contrast, alanine substitution at the same site in JEV NS3 did not increase basal ATPase activity which remained to be stimulated by poly(U). Thus, the mutational effect at His in motif II was different in the HCV and JEV NS3 proteins. Mutagenesis at His-288 of JEV NS3 revealed that His was the most preferable amino acid for ATPase activity and Ala, Gly, Asn, Gln, Ser, or Arg could partly substitute for it. However, any other mutation at His-288 completely disrupted the RNA helicase activity of JEV NS3. The results suggest that Cys-287 and His-288 are essential residues especially for the RNA helicase activity of JEV NS3 and the ATPase and helicase activities are separable enzymatic functions.