Director's Report 5/02 Director's Report to the National Advisory Council on Drug Abuse May, 2002 Index Research Findings Basic Research Behavioral Research Treatment Research and Development Research on AIDS and Other Medical Consequences of Drug Abuse Epidemiology, Etiology and Prevention Research Services Research Intramural Research Program Activities Extramural Policy and Review Activities Congressional Affairs International Activities Meetings and Conferences Media and Education Activities Planned Meetings Publications Staff Highlights Grantee Honors https://archives.drugabuse.gov/DirReports/DirRep502/DirectorRepIndex.html[11/17/16, 10:02:17 PM] Director's Report 5/02 [Office of Director] [First Report Section] Archive Home | Accessibility | Privacy | FOIA (NIH) | Current NIDA Home Page The National Institute on Drug Abuse (NIDA) is part of the National Institutes of Health (NIH) , a component of the U.S. Department of Health and Human Services. Questions? _ See our Contact Information. https://archives.drugabuse.gov/DirReports/DirRep502/DirectorRepIndex.html[11/17/16, 10:02:17 PM] Director's Report 5/02 - Basic Research Director's Report to the National Advisory Council on Drug Abuse May, 2002 Research Findings Basic Research Dopamine Transport The human dopamine transporter (hDAT) serves to regulate the ratio of extracellular to intracellular levels of the neurotransmitter dopamine. Inward transport or influx involves binding of synaptic dopamine at the surface of a neuron, transport across the cell membrane, and release to the intracellular plasma. The process is saturable, and is coupled with the inward transport of sodium and chloride ions. In the reverse or efflux direction, dopamine or related monoamines can be released to the outside of the cell. For kinetic analysis of influx and efflux, useful models of the transporter have proposed an "outward-facing" DAT conformation(s), and an "inward-facing" conformation(s) which would allow external or internal binding of ligands, respectively. One feature of these models is that they might operate in a gated fashion, alternately providing binding sites at the extracellular side of the membrane while blocking the intracellular side, and vice versa. It is presently not known whether the same set of amino acid residues control such transporter conformations of the DAT in both directions, and mutational studies are being extensively carried out to identify the critical residues of dopamine and drugs of abuse. Transport and the DAT are of considerable significance in drug abuse research because monoamines such as amphetamine can be transported, the neurotoxic cation MPP+ can be internalized by transport, and cocaine is known to block or inhibit the uptake of dopamine, by binding to the DAT at one or more sites. Dr. Ulrik Gether and collaborators have previously identified an endogenous high affinity zinc binding site on the DAT which involves the close association in space of histidine at position 193 (extracellular loop 2), histidine 375 (transmembrane helix 7), and glutamate 396 (extracellular loop 4, top of transmembrane helix 8). This zinc binding site, which is largely extracellular, has been shown to act as a noncompetitive inhibitor of dopamine uptake at micromolar zinc concentrations. As an extension of this work, Dr. Gether's research group has now reported that when the intracellular loop 3 residue tyrosine 355 was mutated to alanine, dopamine uptake velocity (Vmax) was reduced to less than 1% of the DAT wild type velocity, and produced a twenty fold increase in the inhibition constant Ki for dopamine binding, all in the absence of zinc. In the presence of ten micromolar zinc, the velocity of uptake was 75% restored. This work was done with COS-1 cells transiently expressing the hDAT. Double mutations, such as mutating both histidine 335 and positions 193, 375, or 396, were not effective in promoting transport of dopamine. The authors have separately shown that the DAT mutated at position 355 appeared to be functional, in that it could be tagged with green fluorescent protein, expressed in HEK- 293 cells, and visualized at the cell surface, and then internalized by activating a protein kinase. It has also been possible to show that the binding of cocaine (which is not transported) was reduced over one hundred fold by the single mutation at position 355, in the absence of zinc, and this reduction was partially reversed in the presence of zinc. The authors have suggested a model in which histidine 355 is needed to stabilize conformations permitting inward transport, with zinc influencing the equilibrium distribution of these conformations. Loland, C.J., Norregaard, L., Litman, T., and Gether, U. Proceedings of the National Academy of Sciences, 99(3), pp. 1683-1688, 2002. Crystal Structure of Biphalin - Multireceptor Opioid Peptide The opioid system plays the most important role in pain signal modulation. This system combines transmembrane G- protein coupled receptors, and their endogenous ligands and opioid peptides released by neurocells. It is widely accepted that there are at least three opioid receptor types, μ, λ, and κ. All opioid receptors have binding sites for benzomorphan alkaloids. Extensive structure-activity studies have shown that recognition of benzomorphan tyramine moiety is a common feature of all opioid receptors. The same tyramine moiety is a part of N-terminal tyrosine, the https://archives.drugabuse.gov/DirReports/DirRep502/DirectorReport1.html[11/17/16, 10:02:21 PM] Director's Report 5/02 - Basic Research active site of endogenous opioid peptides. Large numbers of endogenous opioid peptides, which have been identified, could be divided into groups represented by the endomorphins (large peptide with preference to μ), enkephlins (with affinity with both μ and γ), dynorphins (with selectivity to K), and endomorphins (with selectivity to μ). Opioid analgesic drugs, like morphine, activate opioid receptors that initiate a cascade of events, which results in blocking the pain signal. However, the opioid system is involved in a number of homeostatic neurological and immunological functions. As a result, activation of the opioid system results in a number of side effects, including respiratory depression and dependency. Therefore, one of the most common ways of searching for new opioid analgesic drugs is developing the compounds that have the highest possible selectivity to the receptor and the least unwanted side effect(s). Nevertheless, all opioid receptors are involved in pain transmission modulation. Therefore, the contradictory approach of opioid analgesic development is to search for drugs with affinities to the very broad spectrum of receptors modulating the pain signals. The discovery of biphalin is the best example of the success of using this type of approach. This peptide expresses high affinity to all three opioid receptor types, with some preference for μ receptors. When administered directly into the brain, it has been shown to be more potent than morphine and etorphine at eliciting antinociception (pain relief). Currently biphalin is under intensive preclinical studies. Benzomorphan alkaloid analogues that express different receptor activity, as agonist or antagonist, all possess tyramine moiety in common freeze conformation. This may suggest that opioid peptides, during interaction with receptors, also adopt respective conformation of the tyramine part of N-terminal tyrosine. The presence of other functional groups in opioid peptide analogue and possibility of adopting specific topographical conformation(s) by these groups, determine receptor selectivity and potency. Topographical requirements of different opioid receptors are different even for the same group. Biphalin expresses high affinity to all opioid receptor types. This means that biphalin (i) possesses groups which guarantee high affinity to all types of opioid receptors, (ii) does not possess groups which could negatively interfere with particular receptor subtypes, and (iii) has a peptide chain that is flexible enough to adopt topographical requirements of all opioid receptor types. Flippen-Anderson, J.L., Deschamps, J.R., George, C., Hruby, V.J., Misicka, A., and Lipkowski, A.W. Journal of Peptide Research, 59, pp. 123-133, 2002. Epileptiform Events in CA3 Hippocampus Depressed by Activation of the Opioid-Receptor- like-1 Receptor The CA3 hippocampal region is important in the generation of hippocampal seizures. The opioid-receptor-like-1 (ORL- 1) shares a high sequence homology with opioid receptors and is highly expressed in rat hippocampus. Activation of the receptor robustly depressed spontaneous epileptiform bursting without desensitization, and this was reversed by application of the receptor antagonist. This depressive action is consistent with the general inhibitory nature that occurs as a result of the activation of the ORL-1 receptor as it reduces the spontaneous miniature excitatory post synaptic currents (EPSCs) as well as electro-stimulation induced EPSCs. These observations also indicate that both pre- and post-synaptic mechanisms are involved in the depressive effect of epileptiform activity in CA3. This is interesting as it was thought that in CA1, CA3 and dentate, the ORL-1 receptor had inhibitory postsynaptic actions and seemed to lack the disinhibitory actions. Two membrane currents (channels) activated by the ORL-1 receptor agonist nociceptin were found to mediate the depressive effect of this drug on the epileptiform activity. The relation of the cell