The photoactivatable inhibitor 7-azido-8-iodoketanserin labels the N terminus of the vesicular monoamine transporter from bovine chromaffin granules.
In monoaminergic cells, the hormone or neurotransmitter is concentrated into secretory vesicles by a tetrabenazine- and reserpine-sensitive vesicular monoamine transporter (VMAT), catalyzing a H+/monoamine antiport. Ketanserin is another powerful inhibitor of VMAT that binds to the tetrabenazine binding site. A photoactivatable derivative, 7-azido-8-iodoketanserin (AZIK), labels covalently the transporter from bovine chromaffin granules, VMAT-2. Digestion with endoproteinases V8 or Lys-C, which cleave peptide bonds at acidic or lysine residues, respectively, revealed that the AZIK label is located in a 7 kDa segment of the VMAT-2 polypeptide. The photolabeled chromaffin granule transporter was purified by DEAE and WGA chromatography followed by selective aggregation and size-exclusion HPLC. After treatment by V8 or Lys-C, digestion products were separated by electrophoresis in SDS and sequenced. For both enzymes, the material comigrating with the labeled peptide produced a sequence matching the N terminus of VMAT-2. A K55E mutant of the bovine VMAT-2 cDNA was constructed and expressed in COS-7 cells. The mutant protein exhibited a full VMAT activity and could be labeled by AZIK. However, the formation of the 7 kDa labeled peptide upon Lys-C proteolysis was prevented in the mutant, with a redistribution of the label in higher-molecular mass digestion products. The localization of the label upstream of lysine 55 was confirmed by an immunological and enzymatic analysis. We conclude that the segment 2-55 of bovine VMAT-2, which encompasses the cytosolic N terminus and the first transmembrane segment in the current topological model of the transporter, contains residues involved in the binding of ketanserin and, possibly, tetrabenazine.
Sagné
C
C
CNRS UPR 9071, Institut de Biologie Physico-Chimique, Paris, France.
Isambert
M F
MF
Vandekerckhove
J
J
Henry
J P
JP
Gasnier
B
B
D000313:Adrenal Medulla / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D019556:COS Cells; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D007425:Intracellular Membranes / Q000378:metabolism; D007457:Iodine Radioisotopes; D007650:Ketanserin / Q000031:analogs & derivatives* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D000345:Affinity Labels; D001386:Azides; D017931:DNA Primers; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; C067115:azidoiodoketanserin; D007650:Ketanserin