Opiate drugs have potent analgesic and addictive properties. These drugs interact with receptors that also mediate the response to endogenous opioid peptide ligands. However, the receptors for opioids have eluded definitive molecular characterization. By transient expression in COS cells and screening with an iodinated analog of the opioid peptide enkephalin, a complementary DNA clone encoding a functional delta opioid receptor has been identified. The sequence shows homology to G protein-coupled receptors, in particular the receptors for somatostatin, angiotensin, and interleukin-8.

The analgesic and addictive properties of morphine and other opioid drugs are thought to result from their interaction with mu opioid receptors. Using a delta opioid receptor cDNA as a probe, we have isolated a murine mu opioid receptor cDNA clone (mMOR). Stable expression of mMOR in Chinese hamster ovary cells conferred high binding affinity for mu receptor ligands including morphine and [D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin and low affinity for delta and kappa preferring ligands. Treatment of these cell lines with morphine and other mu agonists inhibited forskolin-induced cAMP accumulation, demonstrating a functional coupling of mMOR to the inhibition of adenylate cyclase. The predicted amino acid sequence of mMOR shares approximately 55% overall amino acid identity with the delta receptor and approximately 97% identity with the recently reported rat mu opioid receptor. Expression of the mu receptor in mouse brain as revealed by in situ hybridization parallels the reported pattern of distribution of mu-selective ligand binding sites. Chromosomal localization (to mouse chromosome 10) and Southern analysis are consistent with a single mu opioid receptor gene in the mouse genome, suggesting that the various pharmacologically distinct forms of the mu receptor arise from alternative splicing, post-translational events, or from a highly divergent gene(s).

Synexin is a calcium-dependent membrane binding protein that not only fuses membranes but also acts as a voltage-dependent calcium channel. We have isolated and sequenced a set ofoverlapping cDNA clones for human synexin. The derived amino acid sequence of synexin reveals strong homology in the C-terminal domain with a previously identified class of calcium-dependent membrane binding proteins. (18,19). cDNA inserts of the three positive recombinants or restriction fragments of the largest cDNA (L4a) were subcloned into M13 for DNA sequencing with M13-or synexinspecific primers (20). Specific oligonucleotide or cDNA probes were used to screen additional cDNA libraries: retina from Jeremy Nathans (Genentech) (Fig. 2); B-cell, adrenal, and lung from Clontech; and fibroblast from H. Okayama (National Institutes of Health) (data not shown) (20).Single Channel Current Measurements. Calcium channel activity of human synexin was measured by methods previously described (refs. 13, 21; Fig. 1 B and C). Briefly, bilayers of phosphatidylserine (PtdSer) or phosphatidylinositol (PtdIns) (Avanti Polar Lipids) were prepared at the tips of patch pipets by a double dip method. Upon forming the bilayer the open tip resistance rose from 15-20 MQ to 3-5 GQl, and channel activity was acquired only after addition of synexin to the bath. The compositions of the various solutions are stated either in the legend to Fig. 1 or in the text.

Reinforcing properties of drugs of abuse are reduced by the coadministration of kappa opioid receptor (KOR) agonists. This effect is related to the inhibition of dopamine (DA) release in the nucleus accumbens (NAc) produced by the acute administration of KOR agonists. The present study was undertaken to investigate the in vivo effect of the repeated administration of KOR agonist on extracellular DA levels in the NAc. Rats were injected once daily with the selective KOR agonist U-69593 (0.16-0.32 mg/kg) or vehicle for 4 days. Microdialysis studies assessing extracellular concentration of DA in the NAc under basal and K(+)-stimulatory conditions were conducted 1 day later. The microdialysis studies revealed that preexposure to U-69593 had no effect on basal extracellular DA levels but significantly augmented the amount of extracellular DA induced by high K(+) compared with vehicle pretreated rats. The D2 receptor agonist quinpirole perfused through the dialysis probe in the NAc, although it produced a significant decrease on basal and K(+)-stimulated DA levels in control rats, it did not decrease significantly either basal or K(+)-stimulated DA levels in U-69593 preexposed rats. Preexposure to U-69593 did not alter the expression of tyrosine hydroxylase or dopamine transporter in the ventral tegmental area. These results show that repeated administration of U-696593 increases the amount of extracellular DA induced by high K in the NAc, an effect that may be related to decreased D2 autoreceptor function. It is suggested that repeated activation of KOR changes the response status of dopaminergic neurons in the NAc.