The results of epitope mapping and limited proteolysis are

The results of epitope mapping and limited proteolysis are also consistent with the results of computer analysis of the sequence. To date, two alternative topological models have been proposed, differing from each other by the orientation of receptor insertion into the lipid bilayer (Fig. 2b and c) [16, 23]. Aydar et al. [23] proposed a topological model of the sigma-1 receptor, where two transmembrane domains of this receptor, TM1 and TM2, are connected in the extracellular space by a loop of about 50 amino phospholipase inhibitor residues, while its N- and C-termini are oriented towards the cytoplasm (see. Fig. 2b) [23]. The C-terminal segment extends for approximately 125 acids, and the N-terminal is relatively short and includes only 10 amino acids. An alternative topological model was proposed by Hayashi and Su; according to them, the sigma-1 receptor is localized in the ER membranes, while its N- and C-termini are facing the ER lumen (see. Fig. 2c) [16]. A di-arginine (R7E/R8E) motif, which is a signal for the retention of the receptor in the ER membranes, is located at the N-terminal of the protein [30,40]. There are currently several known full-length splice variants of the full-length sigma-1 receptor. One of the forms is a variant lacking exon 3 (amino acids 119–149) [41]. Another form that has been described is a truncated sigma-1 receptor the alternative splicing of whose mRNA results in the formation of a premature stop codon (12-kDa variant) [42]. It is known that both forms are incapable of binding ligands. Their functional significance also remains unknown. It has been shown that the isolated transmembrane (amino acids 1–116) and C-terminal (amino acids 116–223) receptor domains also lack the ability to bind ligands, even though some of the receptor\'s functions are preserved (in particular, the ability to activate the Ins3P receptor or the chaperone activity of the C-terminal segment) [16, 43]. Significantly, the isolated C-terminal domain is also localized in the ER membranes, which is consistent with the presence of a third hydrophobic membrane-bound region near SBDLII. NMR spectroscopy showed that residues 198–206 correspond to this membrane-bound region [44]. Recently, Ortega-Roldan et al. determined the secondary structure of the sigma-1 receptor by NMR spectroscopy [44]. The hydrophobic TM2 domain is located around residues 91–107, whereas the amphipathic membrane-bound region is within the range of residues 198–206. The SBDLI and SBDLII segments (see. Fig. 2a) have been identified as alpha-helical. The loop facing the cytosol is formed by several short alpha-helical regions and loop sections. The same group of authors has studied conformational changes occurring in the C-terminal segment (amino acids 112–223) through binding to the ER chaperone protein GRP78/BiP [38], which have been shown to affect virtually all amino acid residues of the sigma-1 receptor. At present, it has been established through site-directed mutagenesis, photoaffinity labeling and molecular simulation techniques that all the domains of a compact receptor-binding site are in one way or another involved in its formation (see. Fig. 2). The first experiments in mapping the active site using site-directed mutagenesis allowed to identify critical residues in the second transmembrane domain for the ligand-binding function of the receptor: S99, Y103, L105 and L106 [45]. Using specific synthetic ligands with photo-reactive labels, it was shown that the binding site is formed by SBDLI residues 91–109 surrounding the critical nitrogen atom detected in all agonists and antagonists specific for the sigma-1 receptor [46-49]. Additionally, SBDLII amino acids 176–194 are involved in the formation of the binding site, surrounding the phenyl ring of the ligand. Chen et al. [49] described in their paper [49] the cocaine binding site in the sigma-1 receptor and, using photosensitive 3-iodo-4-azidococaine and radiosequencing, identified the D188 residue (located in the SBDLII region) as one of the key amino acids involved in the formation of a ligand-binding site.