Opioid receptors have been shown to dimerize or oligomerize among themselves and each other, affecting their functional properties. This study used bioluminescence resonance energy transfer (BRET) between the , ␦, and opioid receptors to study opioid receptor aggregation in transfected human embryonic kidney 293 cells. Titration of receptor levels indicated that all three opioid receptors have a similar affinity to form homo-or hetero-oligomers in combination with any other opioid receptor type. In contrast, none of the opioid receptors formed detectable oligomers with the muscarinic M2 receptor, indicating that interactions among opioid receptors are selective. The formation of opioid receptor dimers, rather than higher order oligomers, is supported by binding kinetics in competition experiments between labeled and unlabeled receptors. Opioid receptor dimerization occurred at physiological temperatures upon receptor biosynthesis, before trafficking to the plasma membrane. Moreover, using BRET, coimmunoprecipitation, receptor binding, and G protein coupling, we demonstrate for the first time functional opioid receptor-opioid receptor heterodimerization. These combined results demonstrate that opioid receptors can undergo homo-and heterodimerization, a process with potential implications for opioid physiology and pharmacology.

Opioid receptors display basal signaling (constitutive, agonistindependent activity), which seems to be regulated by agonist exposure. Whereas agonist pretreatment desensitizes receptors to subsequent agonist stimulation, basal signaling of -opioid receptor (MOR) was shown to increase. Moreover, agonist pretreatment converts the neutral antagonists naloxone and naltrexone into inverse agonists, suppressing basal signaling, whereas analogs with reduced C6-position, e.g., 6␤-naltrexol, remain neutral antagonists at MOR under any condition. This study compares the regulation of basal signaling of MOR, ␦-(DOR), and -(KOR) opioid receptors after pretreatment with morphine or receptor-selective agonists, in transfected human embryonic kidney 293 cell membranes. Moreover, naloxone, naltrexone, and related antagonists were compared for binding potency and effect on basal and agonist-stimulated receptor signaling, measuring guanosine 5Ј-O-(3-[35 S]thio)triphosphate binding. The results demonstrate basal activity for each opioid receptor, which is modulated by pretreatment with agonists. Even closely related opioid antagonists display distinct patterns of neutral and inverse effects before and after agonist pretreatment, including distinct efficacies between naloxone and naltrexone at agonist-pretreated DOR and KOR. Pretreatment with different agonists has varying effects on inverse and neutral activities of some analogs tested. These results demonstrate that antagonist efficacy is context-dependent, possibly accounting for paradoxical pharmacological effects. Activity profiles at the three opioid receptors under different conditions could lead to antagonists with optimal clinical properties in treatment of addiction and adverse opioid effects.

A series of stable mesoporous silica sieves (SBA-15) with different pore sizes (9.8, 7.2, and 5.5 nm) were synthesized using a hydrothermal method. The resulting mesoporous material was then utilized for protein immobilization using myoglobin (Mb) as the target protein. The effects of pore size and adsorption methods on the immobilization efficiency of Mb in a mesoporous material were studied. The SBA-15 with a pore size of 7.2 nm showed the best loading capacity, reaching 413.8 mg/g. The SBA-15 with a pore size of 9.8 nm showed the highest retained catalytic ability (92.36%). The immobilized enzyme was more stable than the free enzyme. After seven consecutive assay cycles, Mb adsorbed by SBA-15 (Mb/SBA-15) and Mb adsorbed by SBA-15 and crosslinked with glutaraldehyde (Mb/G/SBA-15) retained 36.41% and 62.37% of their initial activity, respectively.