Repeated administration of psychostimulants to rodents can lead to behavioral sensitization. Previous studies, using nonspecific opioid receptor (OR) antagonists, revealed that ORs were involved in modulation of behavioral sensitization to methamphetamine (METH). However, the contribution of OR subtypes remains unclear. In the present study, using μ-OR knockout mice, we examined the role of μ-OR in the development of METH sensitization. Mice received daily intraperitoneal injection of drug or saline for 7 consecutive days to initiate sensitization. To express sensitization, animals received one injection of drug (the same as for initiation) or saline on day 11. Animal locomotor activity and stereotypy were monitored during the periods of initiation and expression of sensitization. Also, the concentrations of METH and its active metabolite amphetamine in the blood were measured after single and repeated administrations of METH. METH promoted significant locomotor hyperactivity at low doses and stereotyped behaviors at relative high doses (2.5 mg/kg and above). Repeated administration of METH led to the initiation and expression of behavioral sensitization in wild-type mice. METH-induced behavioral responses were attenuated in the μ-OR knockout mice. Haloperidol (a dopamine receptor antagonist) showed a more potent effect in counteracting METH-induced stereotypy in the μ-OR knockout mice. Saline did not induce behavioral sensitization in either genotype. No significant difference was observed in disposition of METH and amphetamine between the two genotypes. Our study indicated that the μ-opioid system is involved in modulating the development of behavioral sensitization to METH.

Subunit III of the three-subunit catalytic core of cytochrome c oxidase (CcO) contains no metal centers, but it does bind two lipids, within a deep cleft, in binding sites conserved from bacteria to humans. Subunit III binds to subunit I, where it prevents the spontaneous suicide inactivation of CcO by decreasing the probability of side reactions at the heme-Cu O2 reduction site in subunit I. Subunit III prevents suicide inactivation by (1) maintaining adequate rates of proton delivery to the heme-Cu active site and (2) stabilizing the structure of the active site during turnover [Mills and Hosler (2005) Biochemistry 44, 4656]. Here, we first show that mutating several individual residues of the conserved lipid binding sites in subunit III disturbs the subunit I-III interface. Then, two lipid binding site mutants were constructed with an affinity tag on subunit III such that the mutant CcOs could be isolated with 100% subunit III. R226A eliminates an ion pair to the phosphate of the outermost lipid of the cleft, while W59A-F86A disrupts interactions with the fatty acid tails of both lipids. Once these mutant CcOs are placed into soybean phospholipid vesicles, where extensive exchange of bacterial for soybean lipids takes place, it is shown that altering the lipid binding sites mimics a major loss of subunit III, even though subunit III is completely retained, in that suicide inactivation becomes much more probable. The rate of proton delivery to the active site remains rapid, ruling out slow proton uptake as the primary reason for increased suicide inactivation upon alteration of the lipid binding sites. We conclude that altering the lipid binding sites of subunit III may promote side reactions leading to suicide inactivation by allowing greater movement to occur in and around the O2 reduction site of subunit I during the catalytic cycle.