χ-conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter

The interactions of chi-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [H-3] norepinephrine (K-i 1.89 muM) than [H-3] dopamine (K-i 4.33 muM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA ( to 7 muM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [H-3] norepinephrine uptake for three mutations ( in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine.

Asymmetric contribution of α and β subunits to the activation of αβ heteromeric glycine receptors

This study investigated the role of beta subunits in the activation of alphabeta heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in alpha and beta subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the alpha subunit M2-M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the beta subunit. Furthermore, although the alpha subunit M2-M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding beta subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between alpha and beta subunit M2-M3 loops. In addition, a threonine-->leucine mutation at the 9' position in the beta subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR beta subunit is involved in receptor gating. In conclusion, this study demonstrates that beta subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the alpha subunit.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.