Melittin stimulates fatty acid release through non-phospholipase-mediated mechanisms and interacts with the dopamine transporter and other membrane-spanning proteins.
Phospholipase A(2) releases the fatty acid arachidonic acid from membrane phospholipids. We used the purported phospholipase A(2) stimulator, melittin, to examine the effects of endogenous arachidonic acid signaling on dopamine transporter function and trafficking. In HEK-293 cells stably transfected with the dopamine transporter, melittin reduced uptake of [((3))H]dopamine. Additionally, measurements of fatty acid content demonstrated a melittin-induced release of membrane-incorporated arachidonic acid, but inhibitors of phospholipase C, phospholipase D, and phospholipase A(2) did not prevent the release. Subsequent experiments measuring [(125)I]RTI-55 binding to the dopamine transporter demonstrated a direct interaction of melittin, or a melittin-activated endogenous compound, with the transporter to inhibit antagonist binding. This effect was not specific to the dopamine transporter, as [(3)H]spiperone binding to the recombinant dopamine D(2) receptor was also inhibited by melittin treatment. Finally, melittin stimulated an increase in internalization of the dopamine transporter, and this effect was blocked by pretreatment with cocaine. Thus, melittin acts through multiple mechanisms to regulate cellular activity, including release of membrane-incorporated fatty acids and interaction with the dopamine transporter.
Keith
Dove J
DJ
Research Service, VA Medical Center, and Graduate Program in Neuroscience, Portland, OR 97239, USA.
Eshleman
Amy J
AJ
Janowsky
Aaron
A
D016718:Arachidonic Acid / Q000378:metabolism*; D001692:Biological Transport; D003042:Cocaine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D004347:Drug Interactions; D020536:Enzyme Activators / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008555:Melitten / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D064801:Phospholipase A2 Inhibitors; D010739:Phospholipase D / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D054467:Phospholipases A2 / Q000378:metabolism; D021381:Protein Transport; D010738:Type C Phospholipases / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D020536:Enzyme Activators; D008565:Membrane Proteins; D064801:Phospholipase A2 Inhibitors; D008555:Melitten; D016718:Arachidonic Acid; D010740:Phospholipases; D054467:Phospholipases A2; D010738:Type C Phospholipases; D010739:Phospholipase D; D003042:Cocaine; D004298:Dopamine