The Photoactivatable Inhibitor 7-Azido-8-iodoketanserin Labels the N Terminus of the Vesicular Monoamine Transporter from Bovine Chromaffin Granules † Corinne Sagne ´, ‡ Marie-Franc ¸ oise Isambert, ‡ Joe ¨l Vandekerckhove, § Jean-Pierre Henry, ‡ and Bruno Gasnier* ,‡ CNRS UPR 9071, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, F-75005 Paris, France, and Flanders InteruniVersity Institute of Biotechnology, Department of Medical Protein Chemistry, UniVersiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium ReceiVed September 17, 1996; ReVised Manuscript ReceiVed January 6, 1997 X ABSTRACT: 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. Non-peptide neurotransmitters are synthesized in the cytoplasm and concentrated in synaptic vesicles or secretory granules, prior to their release by exocytosis. The vesicular uptake involves an inwardly directed ATP-dependent H + pump and a membrane transporter, which catalyzes a H + / neurotransmitter antiport (Schuldiner et al., 1995). The mammalian vesicular monoamine transporter (VMAT), 1 which catalyzes the vesicular uptake of dopamine, norepi- nephrine, epinephrine, serotonin, and histamine in a variety of cells, has recently been characterized by cDNA expression cloning (Erickson et al., 1992; Liu et al., 1992). Two isoforms sharing about 60% amino acid identity, now designated VMAT-1 and VMAT-2, were identified. The two isoforms, which are generally expressed in distinct cells (Peter et al., 1995; Weihe et al., 1994), exhibit some differences in their functional properties (Erickson et al., 1996; Peter et al., 1994). VMAT-2 has a higher affinity than VMAT-1 for monoamine substrates, in particular for hista- mine, and a higher sensitivity to some inhibitors, in particular to tetrabenazine (TBZ). This latter difference is dramatic for the human clones, since no sensitivity to TBZ could be detected for human VMAT-1 (Erickson et al., 1996). Phenotypic selection in the nematode Caenorhabditis elegans allowed the molecular cloning of a highly related vesicular acetylcholine transporter from different species (Alfonso et al., 1993; Erickson et al., 1994; Roghani et al., 1994; Varoqui et al., 1994). The chromaffin granule from bovine adrenal medulla is an abundant natural source of VMAT, which has provided most of our knowledge on the bioenergetics and biochemistry of vesicular neurotransmitter transporters (Schuldiner et al., 1995). The bovine chromaffin granule transporter has been purified (Isambert et al., 1992; Sagne ´ et al., 1996; Stern- Bach et al., 1990; Vincent & Near, 1991) and shown to correspond to the bovine VMAT-2 isoform by amino acid sequencing (Howell et al., 1994; Krejci et al., 1993; Stern- Bach et al., 1992). The study of VMAT benefited also from the existence of two high-affinity inhibitors, reserpine and TBZ (Henry & Scherman, 1989). The two inhibitors differ in their sensitivity to the proton electrochemical gradient and are thought to bind to distinct sites on VMAT (Deupree & Weaver, 1984; Scherman & Henry, 1984; Weaver & Deu- † This work was supported by the Centre National de la Recherche Scientifique (UPR 9071), the European Union BIOMED-1 Program (Contract BMH1-CT93-1110 to J.-P.H.), and the Ministe `re de la Recherche et de la Technologie (fellowship to C.S.). J.V. was supported by EU Grant CHRX-CT94-0430. * Author to whom correspondence should be addressed. Fax: 33- 1-40468331. E-mail: gasnier@ibpc.fr. ‡ CNRS UPR 9071. § Universiteit Gent. X Abstract published in AdVance ACS Abstracts, March 1, 1997. 1 Abbreviations: [ 125 I]AZIK, 7-azido-8-[ 125 I]iodoketanserin [7-azido- 8-iodo-3-[2-[4-(fluorobenzoyl)-1-piperidinyl]ethyl]-2,4(1H,3H)-quinazo- linedione]; GST, glutathione S-transferase; 5-HT, 5-hydroxytryptamine; PVDF, poly(vinylidene difluoride); TBZ, tetrabenazine (2-oxo-3- isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11b(H)-benzo[a]quino- lizine); [ 3 H]TBZOH, [2- 3 H]dihydrotetrabenazine; Tricine, N-[tris(hy- droxymethyl)methyl]glycine; Tris, tris(hydroxymethyl)aminomethane; VMAT, vesicular monoamine transporter; WGA, wheat germ ag- glutinin. 3345 Biochemistry 1997, 36, 3345-3352 S0006-2960(96)02343-4 CCC: $14.00 © 1997 American Chemical Society