Distinct Regulation of σ ₁ Receptor Multimerization by Its Agonists and Antagonists in Transfected Cells and Rat Liver Membranes

Extensive studies have shown that the σ ₁ receptor (σ ₁R) interacts with and modulates the activity of multiple proteins with important biological functions. Recent crystal structures of σ ₁R as a homotrimer differ from a dimer-tetramer model postulated earlier. It remains inconclusive whether ligand binding regulates σ ₁R oligomerization. Here, novel nondenaturing gel methods and mutational analysis were used to examine σ ₁R oligomerization. In transfected cells, σ ₁R exhibited as multimers, dimers, and monomers. Overall, σ ₁R agonists decreased, whereas σ ₁R antagonists increased σ ₁R multimers, suggesting that agonists and antagonists differentially affect the stability of σ ₁R multimers. Endogenous σ ₁R in rat liver membranes also showed similar regulation of oligomerization as in cells. Mutations at key residues lining the trimerization interface (Arg119, Asp195, Phe191, Trp136, and Gly91) abolished multimerization without disrupting dimerization. Intriguingly, truncation of the N terminus reduced σ ₁R to apparent monomer. These results demonstrate that multiple domains play crucial roles in coordinating high-order quaternary organization of σ ₁R. The E102Q σ ₁R mutant implicated in juvenile amyotrophic lateral sclerosis formed dimers only, suggesting that dysregulation of σ ₁R multimeric assembly may impair its function. Interestingly, oligomerization of σ ₁R was pH-dependent and correlated with changes in [³H](+)-pentazocine binding affinity and B_max Combined with mutational analysis, it is reasoned that σ ₁R multimers possess high-affinity and high-capacity [³H](+)-pentazocine binding, whereas monomers likely lack binding. These results suggest that σ ₁R may exist in interconvertible oligomeric states in a dynamic equilibrium. Further exploration of ligand-regulated σ ₁R multimerization may provide novel approaches to modulate the function of σ ₁R and its interacting proteins. SIGNIFICANCE STATEMENT: The σ ₁ receptor (σ ₁R) modulates the activities of various partner proteins. Recently, crystal structures of σ ₁R were elucidated as homotrimers. This study used novel nondenaturing gel methods to examine σ₁R oligomerization in transfected cells and rat liver membranes. Overall, agonist binding decreased, whereas antagonist binding increased σ ₁R multimers, which comprised trimers and larger units. σ ₁R multimers were shown to bind [³H](+)-pentazocine with high affinity and high capacity. Furthermore, mutational analysis revealed a crucial role of its N-terminal domain in σ ₁R multimerization.