5] Noncovalent and covalent labeling of vesicular monoamine transporter with tetrabenazine and ketanserin derivatives: Purification of photolabeled protein

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The vesicular monoamine transporter (VMAT) accumulates monoamines inside secretory granules or synaptic vesicles by catalyzing an H+/ monoamine antiport. Monoamine uptake is coupled to the generation of a proton electrochemical gradient by an ATP-dependent H + pump of the vacuolar type. The uptake is extremely efficient and in the adrenal medulla chromaffin cells, the monoamine gradient between the cytosol and the secretory granules has been estimated to five orders of magnitude. This chapter discusses noncovalent and covalent labeling of vesicular monoamine transporter with tetrabenazine and ketanserin derivatives protein. The vesicular monoamine transporter from various origins is cloned and expressed in various cell types. Two isoforms, VMAT1 and VMAT2, are identified; they have different localizations and slightly different properties. Two different binding sites are also identified: the first one binds reserpine, whereas the second one binds tetrabenazine (TBZ) and ketanserin (KET). The H+ electrochemical gradient generated in the presence of ATP greatly accelerates reserpine binding, but has no effect on TBZ and KET binding.