Subject(s)
Amphetamine/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Dopamine Plasma Membrane Transport Proteins/metabolism , Locomotion/drug effects , Amphetamine/antagonists & inhibitors , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Dopamine Plasma Membrane Transport Proteins/genetics , Drosophila , Methamphetamine/pharmacology , Mutation , Peptides/pharmacology , Phosphorylation/drug effectsABSTRACT
The dopamine transporter (DAT) is the primary molecular target responsible for the rewarding properties of the psychostimulants amphetamine (AMPH) and cocaine. AMPH increases extracellular dopamine (DA) by promoting its nonexocytotic release via DAT-mediated efflux. Previous studies in heterologous cells have shown that phosphorylation of the amino terminus of DAT is required for AMPH-induced DA efflux but not for DA uptake. However, the identity of many of the modulatory proteins and the molecular mechanisms that coordinate efflux and the ensuing behavioral effects remain poorly defined. Here, we establish a robust assay for AMPH-induced hyperlocomotion in Drosophila melanogaster larvae. Using a variety of genetic and pharmacological approaches, we demonstrate that this behavioral response is dependent on DA and on DAT and its phosphorylation. We also show that methylphenidate (MPH), which competitively inhibits DA uptake but does not induce DAT-mediated DA efflux, also leads to DAT-dependent hyperlocomotion, but this response is independent of DAT phosphorylation. Moreover, we demonstrate that the membrane raft protein Flotillin-1 is required for AMPH-induced, but not MPH-induced, hyperlocomotion. These results are the first evidence of a role for a raft protein in an AMPH-mediated behavior. Thus, using our assay we are able to translate molecular and cellular findings to a behavioral level and to differentiate in vivo the distinct mechanisms of two psychostimulants.
Subject(s)
Amphetamine/pharmacology , Central Nervous System Stimulants/pharmacology , Dopaminergic Neurons/drug effects , Locomotion/drug effects , Membrane Proteins/drug effects , Animals , Dopamine Plasma Membrane Transport Proteins/genetics , Dopamine Plasma Membrane Transport Proteins/metabolism , Drosophila , Membrane Proteins/genetics , Methylphenidate/pharmacology , Mutation , PhosphorylationABSTRACT
S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) is an important enzyme in the trans-sulphuration pathway, mediating the conversion of S-adenosyl-L-homocysteine to adenosine and L-homocysteine. We have identified a cDNA clone from Xenopus laevis, encoding a protein of 433 aa, which is highly conserved with S-Adenosyl-L-homocysteine hydrolases (Adohcyases) from other species. Expression of Adohcyase mRNA in X.laevis tadpoles is detectable from developmental Stage 27 onwards. Phylogenetic analysis of available Adohcyase sequences indicates that species cluster essentially as predicted from morphological data. Furthermore, we estimate that S-adenosyl-L-homocysteine hydrolase is evolving very slowly, almost 10 times slower than the average rate.
