title: Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3.
abstract: Organic cation transporter 3 (OCT3, SLC22A3) is a polyspecific, facilitative transporter expressed in astrocytes and in placental, intestinal, and blood-brain barrier epithelia, and thus elucidating the molecular mechanisms underlying OCT3 substrate recognition is critical for the rational design of drugs targeting these tissues. The pharmacology of OCT3 is distinct from that of other OCTs, and here we investigated the role of a hydrophobic cavity tucked within the translocation pathway in OCT3 transport properties. Replacement of an absolutely conserved Asp by charge reversal (D478E), neutralization (D478N), or even exchange (D478E) abolished MPP(+) uptake, demonstrating this residue to be obligatory for OCT3-mediated transport. Mutations at non-conserved residues lining the putative binding pocket of OCT3 to the corresponding residue in OCT1 (L166F, F450L, and E451Q) reduced the rate of MPP(+) transport, but recapitulated the higher sensitivity pharmacological profile of OCT1. Thus, interactions of natural polyamines (putrescine, spermidine, spermine) and polyamine-like potent OCT1 blockers (1,10-diaminodecane, decamethonium, bistriethylaminodecane, and 1,10-bisquinuclidinedecane) with wild-type OCT3 were weak, but were significantly potentiated in the mutant OCT3s. Conversely, a reciprocal mutation in OCT1 (F161L) shifted the polyamine-sensitivity phenotype toward that of OCT3. Further analysis indicated that OCT1 and OCT3 can recognize essentially the same substrates, but the strength of substrate-transporter interactions is weaker in OCT3, as informed by the distinct makeup of the hydrophobic cleft. The residues identified here are key contributors to both the observed differences between OCT3 and OCT1 and to the mechanisms of substrate recognition by OCTs in general.
authors.affiliation: From the Department of Cell Biology and Physiology, and the Center for the Investigation of Membrane Excitability Diseases (CIMED), Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Dan C
authors.initials: DC
authors.lastname: Li
authors.affiliation: From the Department of Cell Biology and Physiology, and the Center for the Investigation of Membrane Excitability Diseases (CIMED), Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Colin G
authors.initials: CG
authors.lastname: Nichols
authors.affiliation: From the Department of Cell Biology and Physiology, and the Center for the Investigation of Membrane Excitability Diseases (CIMED), Washington University School of Medicine, St. Louis, Missouri 63110 msala@wustl.edu.
authors.forename: Monica
authors.initials: M
authors.lastname: Sala-Rabanal
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003959:Diamines / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009154:Mutation; D027701:Organic Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics; D027702:Organic Cation Transporter 1 / Q000737:chemistry / Q000235:genetics; D011073:Polyamines / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011700:Putrescine / Q000737:chemistry; D051381:Rats; D013095:Spermidine / Q000737:chemistry; D000099254:Organic Cation Transporter 3
chemical_list: D003959:Diamines; D027701:Organic Cation Transport Proteins; D027702:Organic Cation Transporter 1; D011073:Polyamines; D011700:Putrescine; D013095:Spermidine; D000099254:Organic Cation Transporter 3; C065351:1,10-diaminodecane
keywords: OCT3; SLC22A3; membrane transport; mutagenesis; organic cation transporter; pharmacology; polyamine; rational drug design; structure-function