TY - JOUR

T1 - An intracellular interaction network regulates conformational transitions in the dopamine transporter.

AU - Kniazeff, Julie

AU - Shi, Lei

AU - Løland, Claus Juul

AU - Javitch, Jonathan A

AU - Weinstein, Harel

AU - Gether, Ulrik

N1 - Keywords: Amino Acid Sequence; Animals; COS Cells; Cercopithecus aethiops; Cytoplasm; Dopamine Plasma Membrane Transport Proteins; Gene Expression Regulation; Humans; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Neurotransmitter Agents; Protein Conformation; Sequence Homology, Amino Acid; Sodium

PY - 2008

Y1 - 2008

N2 - Neurotransmitter:sodium symporters (NSS)(1) mediate sodium-dependent reuptake of neurotransmitters from the synaptic cleft and are targets for many psychoactive drugs. The crystal structure of the prokaryotic NSS protein, LeuT, was recently solved at high resolution; however, the mechanistic details of regulation of the permeation pathway in this class of proteins remain unknown. Here we combine computational modeling and experimental probing in the dopamine transporter (DAT) to demonstrate the functional importance of a conserved intracellular interaction network. Our data suggest that a salt bridge between Arg-60 in the N terminus close to the cytoplasmic end of transmembrane segment (TM) 1 and Asp-436 at the cytoplasmic end of TM8 is stabilized by a cation-pi interaction between Arg-60 and Tyr-335 at the cytoplasmic end of TM6. Computational probing illustrates how the interactions may determine the flexibility of the permeation pathway, and mutagenesis within the network and results from assays of transport, as well as the state-dependent accessibility of a substituted cysteine in TM3, support the role of this network in regulating access between the substrate binding site and the intracellular milieu. The mechanism that emerges from these findings may be unique to the NSS family, where the local disruption of ionic interactions modulates the transition of the transporter between the outward- and inward-facing conformations.

AB - Neurotransmitter:sodium symporters (NSS)(1) mediate sodium-dependent reuptake of neurotransmitters from the synaptic cleft and are targets for many psychoactive drugs. The crystal structure of the prokaryotic NSS protein, LeuT, was recently solved at high resolution; however, the mechanistic details of regulation of the permeation pathway in this class of proteins remain unknown. Here we combine computational modeling and experimental probing in the dopamine transporter (DAT) to demonstrate the functional importance of a conserved intracellular interaction network. Our data suggest that a salt bridge between Arg-60 in the N terminus close to the cytoplasmic end of transmembrane segment (TM) 1 and Asp-436 at the cytoplasmic end of TM8 is stabilized by a cation-pi interaction between Arg-60 and Tyr-335 at the cytoplasmic end of TM6. Computational probing illustrates how the interactions may determine the flexibility of the permeation pathway, and mutagenesis within the network and results from assays of transport, as well as the state-dependent accessibility of a substituted cysteine in TM3, support the role of this network in regulating access between the substrate binding site and the intracellular milieu. The mechanism that emerges from these findings may be unique to the NSS family, where the local disruption of ionic interactions modulates the transition of the transporter between the outward- and inward-facing conformations.

U2 - 10.1074/jbc.M800475200

DO - 10.1074/jbc.M800475200

M3 - Journal article

C2 - 18426798

VL - 283

SP - 17691

EP - 17701

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 25

ER -