Eric L. Barker

High Affinity Recognition of Serotonin Transporter Antagonists Defined by Species-scanning Mutagenesis AN AROMATIC RESIDUE IN TRANSMEMBRANE DOMAIN I DICTATES SPECIES-SELECTIVE RECOGNITION OF CITALOPRAM AND MAZINDOL

Human and Drosophila melanogasterserotonin (5-HT) transporters (SERTs) exhibit similar 5-HT transport kinetics and can be distinguished pharmacologically by many, but not all, biogenic amine transporter antagonists. By using human andDrosophila SERT chimeras, major determinants of potencies of two transporter antagonists, mazindol and citalopram, were tracked to the amino-terminal domains encompassing transmembrane domains I and II. Species-scanning mutagenesis, whereby amino acid substitutions are made switching residues from one species to another, was employed on the eight amino acids that differ between human andDrosophila SERTs in this region, and antagonist potencies were reassessed in 5-HT uptake assays. A single mutation in transmembrane domain I of human SERT, Y95F, shifted both citalopram and mazindol to Drosophila SERT-like potencies. Strikingly, these potency changes were in opposite directions suggesting Tyr95 contributes both positive and negative determinants of antagonist potency. To gain insight into how the Y95F mutant might influence mazindol potency, we determined how structural variants of mazindol responded to the mutation. Our studies demonstrate the importance of the hydroxyl group on the heterocyclic nucleus of mazindol for maintaining species-selective recognition of mazindol and suggest that transmembrane domain I participates in the formation of antagonist-binding sites for amine transporters.

Inactivation and Biotransformation of the Endogenous Cannabinoids Anandamide and 2-Arachidonoylglycerol

The cannabinoid field is currently an active research area. Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the most characterized endogenous cannabinoids (also known as endocannabinoids). These neuromodulators have been implicated in various physiologically relevant phenomena, including mood (Witkin et al., 2005), the immune response (Ashton, 2007), appetite (Kirkham and Tucci, 2006), reproduction (Wang et al., 2006), spasticity (Pertwee, 2002), and pain (Hohmann and Suplita, 2006). Pharmacological manipulation of AEA and 2-AG signaling should prove to have significant therapeutic applications in disorders linked to endocannabinoid signaling. One way to alter endocannabinoid signaling is to regulate the events responsible for termination of the endocannabinoid signal-cellular uptake and metabolism. However, to pharmacologically exploit AEA and/or 2-AG signaling in this way, we must first gain a better understanding of the proteins and mechanisms governing these processes. This review serves as an introduction to the endocannabinoid system with an emphasis on the proteins and events responsible for the termination of AEA and 2-AG signaling.

Comparative Molecular Field Analysis Using Selectivity Fields Reveals Residues in the Third Transmembrane Helix of the Serotonin Transporter Associated with Substrate and Antagonist Recognition

The human serotonin transporter (hSERT) regulates the spatial and temporal actions of serotonin (5-HT) neurotransmission by removing 5-HT from the synapse. Previous studies have identified residues in the third transmembrane helix (TMH) that may be important for substrate translocation or antagonist recognition. We identified hSERT residues in TMH III that are divergent from Drosophila SERT and used species-scanning mutagenesis to generate reciprocal mutants. Transport inhibition assays suggest that the potency of substituted amphetamines was decreased for the hSERT mutants A169D, I172M, and S174M. In addition, there was a loss of potency for several antidepressants and 3-phenyltropane analogs for the I172M mutant. These results suggest that residues in TMH III may contribute to antagonist recognition. We carried out comparative molecular field analyses using selectivity fields to directly visualize the mutation-induced effects of antagonist potency for antidepressants, 3-phenyltropane analogs, and amphetamines. The hSERT I172M selectivity field analysis for the 3-phenyltropane analogs revealed that electrostatic interactions resulted in decreased potency. The amphetamine and antidepressant selectivity field analyses reveal the observed decreases in potencies for the hSERT I172M mutant are due to a change in tertiary structure of the hSERT protein and are not due to disruption of a direct binding site. Finally, the hSERT mutant A169D displayed altered kinetics for sodium binding, indicating that this residue may lie near the putative sodium binding site. A SERT homology model developed from the Aquifex aeolicus leucine transporter structure provides a structural context for further interpreting the results of the TMH III mutations.

RNA interference-mediated knockdown of dynamin 2 reduces endocannabinoid uptake into neuronal dCAD cells.

The precise mechanism by which the cellular uptake of the endocannabinoid anandamide (AEA) occurs has been the source of much debate. In the current study, we show that neuronal differentiated CAD (dCAD) cells accumulate anandamide by a process that is inhibited in a dose-dependent manner by N-(4-hydroxyphenyl)arachidonylamide (AM404). We also show that dCAD cells express functional fatty acid amide hydrolase, the enzyme primarily responsible for anandamide metabolism. Previous data from our laboratory indicated that anandamide uptake occurs by a caveolae-related endocytic mechanism in RBL-2H3 cells. In the current study, we show that anandamide uptake by dCAD cells may also occur by an endocytic process that is associated with detergent-resistant membrane microdomains or lipid rafts. Nystatin and progesterone pretreatment of dCAD cells significantly inhibited anandamide accumulation. Furthermore, RNA interference (RNAi)-mediated knockdown of dynamin 2, a protein involved in endocytosis, blocked the internalization of the fluorescently labeled anandamide analog SKM 4-45-1 ([3′,6′-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthen-5-yl]-2-[[1-oxo-5Z,8Z,11Z,14Z-eicosatetraenyl]amino]ethyl ester carbamic acid). RNAi-mediated knockdown of the β2 subunit of the clathrin-associated activator protein 2 complex had no effect on SKM 4-45-1 internalization. We were surprised to find that dynamin 2 knockdown in dCAD cells did not affect [3H]AEA uptake. However, dynamin 2 knockdown caused a significant increase in the overall levels of intact [3H]AEA associated with the cells, suggesting that trafficking of [3H]AEA to FAAH had been disrupted. This finding may be the result of an accumulation of the anandamide carrier protein in detergent-resistant membranes after dynamin 2 knockdown. Our studies provide evidence that the cellular uptake of anandamide may occur by a dynamin 2-dependent, caveolae-related endocytic process in dCAD cells.

Structural determinants of neurotransmitter transport using cross-species chimeras: studies on serotonin transporter.

Publisher Summary This chapter describes the formation and analysis of cross-species chimeras. It illustrates how the species variants of serotonin (5-hydroxytryptamine (5-HT)) transporter (SERT) have served as an important guide for molecular structure-function studies. The ultimate goal of structure–function studies of SERT and related neurotransmitter transporters is to gain a better understanding of transport mechanisms to provide insight into how the proteins may be altered in disease states and regulated by endogenous or therapeutic agents. SERTs are most closely related to the transporters for the monoamine transmitters norepinephrine and dopamine (DATs), all of which are modeled to possess 12 putative transmembrane domains with cytoplasmic amino and carboxyl tails. SERTs and DATs are of particular interest as are implicated in multiple psychiatric diseases.

Structural Analysis of the Extracellular Entrance to the Serotonin Transporter Permeation Pathway

Neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. These transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. A high resolution crystal structure for the monoamine transporters has yet to be solved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuTAa) from Aquifex aeolicus. The objective of the present studies is to identify the structural determinants forming the entrance to the substrate permeation pathway based on predictions from the SERT homology model. Using the substituted cysteine accessibility method, we identified residues predicted to reside at the entrance to the substrate permeation pathway that were reactive with methanethiosulfonate (MTS) reagents. Of these residues, Gln332 in transmembrane helix (TMH) VI was protected against MTS inactivation in the presence of serotonin. Surprisingly, the reactivity of Gln332 to MTS reagents was enhanced in the presence of cocaine. Bifunctional MTS cross-linkers also were used to examine the distances between helices predicted to form the entrance into the substrate and ion permeation pathway. Our studies suggest that substrate and ligand binding may induce conformational shifts in TMH I and/or VI, providing new opportunities to refine existing homology models of SERT and related monoamine transporters.

Phosphoinositide hydrolysis linked 5-HT2 receptors in fibroblasts from choroid plexus

A serotonin (5-HT)-mediated phosphoinositide hydrolysis response was characterized in fibroblasts cultured from rabbit choroid plexus. 5-HT elicited a maximum 8-fold increase in [3H]inositol-phosphate ([3H]IP) formation, while the partial agonists, (+)-lysergic acid diethylamide and (-)-1-(4-bromo-2,5-dimethyoxyphenyl)-2-aminopropane caused 2- and 5-fold increases, respectively. Mianserin, ketanserin, and spiperone were equipotent at blocking the 5-HT-mediated response. Thus, agonist and antagonist profiles indicate interactions with 5-HT2 receptors.

VOLTAMMETRIC APPROACHES TO KINETICS AND MECHANISM OF THE NOREPINEPHRINE TRANSPORTER

Publisher Summary Rotating-disk electrode voltammetry (RDEV) has been used to determine kinetic arameters of dopamine (DA) and norepinephrine (NE) at the hNET in LLC-NET cells. As uptake occurs, the decreasing current is recorded for 6 min as a new steady-state is approached. To induce efflux, 6 μ l of unbuffered electrolyte as a control or 1, 10, or 100 μ M of selected substrate is added to the suspension, and the efflux of DA is monitored. At first, there is little l-amphetamine relative to the high concentration of DA on the inside of the cells to compete for outward transport. However, as it accumulates intracellularly, it competes with DA for binding to the empty transporter and DA efflux decreases. At the same time, DA concentration in the medium increases, causing an increase in the inward flux of DA. Amphetamine is known to inhibit the uptake of norepinephrine across the neuronal membrane and participate in the transmitters release through the transporter. It was hypothesized that amphetamine-induced release was through an exchange diffusion mechanism. Because of the highly lipophilic nature of amphetamine, it is also capable of entering the cell by diffusion, complicating the analysis. The effect of using methylphenidate as an effluxing agent was also examined. Methylphenidate, as with d -amphetamine, has been used to treat attention deficit disorder with hyperactivity. Its mode of action is believed to be similar to that of cocaine in that it inhibits the DA transporter.

4-Aminocyclopentane-1,3-diols as Platforms for Diversity: Synthesis of Anandamide Analogs

Starting from cyclopentadiene, two racemic mixtures of 4-aminocyclopentane-1,3-diols were prepared in 8 steps and characterized. Structure determination proved the anticipated trans-orientation of the two oxygen atoms with respect to the plane of the ring. The fragment-like new compounds are small and hydrophilic, devoid of rotatable bonds, and offer stereochemically defined attachment points for substituents. Thus, these platforms for diversity are suitable starting points for the construction of combinatorial libraries of lead-like 4-amidocyclopentane-1,3-diols or natural product analogs. As a proof of concept, cyclopentanoid anandamide analogs were prepared using these molecular platforms and evaluated as tools for the investigation of unresolved issues in the molecular biology of anandamide.

Dr Roger P Maickel, 1933–2006

Dr Roger P Maickel, 73, of Lafayette, IN, died on Tuesday, December 26, 2006. The Department of Medicinal Chemistry and Molecular Pharmacology as well as all of his colleagues in the Purdue University School of Pharmacy and Pharmaceutical Sciences are deeply saddened by his death. Dr Maickel was a dear friend, colleague, mentor, and the former Department Head. Born on September 8, 1933, in Floral Park, Long Island, NY, he was the son of the late Philip V and Margaret M Rose Maickel. He lived in Washington, DC, and Bloomington, IN before moving to Lafayette, IN. He graduated from Chaminade High School, received his bachelor’s degree in 1954 at Manhattan College, master’s degree in 1957, and his doctorate in 1960 from Georgetown University. Dr Maickel worked for the National Institute of Health as a research scientist from 1956 to 1965, was professor of pharmacology in the medical sciences program at Indiana University from 1965 to 1977, was department head of pharmacology and toxicology at Purdue University from 1977 to 1983, returning as professor at Purdue to continue his research from 1983 to 1999. He became professor emeritus of pharmacology and toxicology in 1999. Dr Maickel received the National Institute of Health Research Development Award 1969–1974 and the NASA Life Scientist Award 1972–1975, was a guest lecturer for the DEA–US Department of Justice for 15 years, served on the US Product Safety Commission 1979–1981, was a consultant to the FDA from 1987 to 1990, served 10 years as a member of the Indiana Controlled Substances Advisory Committee, and co-authored numerous scientific papers and contributed to numerous text books. He was a member of St Thomas Aquinas Center, Catholic Academy of Sciences USA, Knight of the Equestrian Order of the Holy Sepulchre of Jerusalem, American Society for Clinical Pharmacology and Therapeutics, American Institute of Chemist, Federation of American Societies for Experimental Biology, Association for Assessment and Accreditation of Laboratory Animal Care, Collegium Internationale Neuro-Psychopharmacologicum, American Council on Science and Health, Royal Society of Chemistry, Purdue University Presidents Council, Purdue Reamer Club, and John Purdue Club. Dr Maickel was accepted into the American College of Neuropsychopharmacology in 1970 and was a Fellow Emeritus at the time of his death. He enjoyed golf, trains, reading, travel, gardening, photography, and college athletics. Many of his colleagues enjoyed the ‘fruits of his labors’ each summer as he often shared his varieties of hot peppers and tomatoes. He married Lois Pivonka on September 8, 1956, in Ellinwood, KS. He leaves behind wife, two daughters, Nancy Maickel Ward (husband: Michael) of Lebanon, IN and Carolyn Maickel Anderson of Gurnee, IL, and six grandchildren, Matthew, Brian, and Julie Ward and Molly, Erik, and Clark Anderson. Memorials can be made to the Purdue Foundation (please note in the Memo, Maickel Memorial Fund) and sent to Linda Yelton, Development Office, Purdue University School of Pharmacy, 575 Stadium Mall Drive, West Lafayette, IN 47907.