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Involvement of threonine 258 and serine 259 motif in amphetamine‐induced norepinephrine transporter endocytosis
Author(s) -
Annamalai Balasubramaniam,
Mannangatti Padmanabhan,
Arapulisamy Obulakshmi,
Ramamoorthy Sammanda,
Jayanthi Lankupalle D.
Publication year - 2010
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2010.06898.x
Subject(s) - endocytosis , norepinephrine transporter , chemistry , protein kinase c , microbiology and biotechnology , protein kinase a , phosphorylation , transporter , biology , biochemistry , cell , gene
J. Neurochem. (2010) 115 , 23–35. Abstract d ‐Amphetamine (AMPH) down‐regulates the norepinephrine transporter (NET), although the exact trafficking pathways altered and motifs involved are not known. Therefore, we examined the cellular and molecular mechanisms involved in AMPH‐induced NET regulation in human placental trophoblast cells expressing the wild‐type (WT)‐hNET and the hNET double mutant (DM)‐bearing protein kinase C (PKC)‐resistant T258A + S259A motif. NET function and surface expression were significantly reduced in cells expressing WT‐hNET but not in cells expressing hNET‐DM following AMPH treatment. AMPH inhibited plasma membrane recycling of both WT‐hNET and hNET‐DM. In contrast, AMPH stimulated endocytosis of WT‐hNET, and did not affect hNET‐DM endocytosis. Although PKC or calcium/calmodulin‐ dependent kinase‐II (CaMKII) inhibition or depletion of calcium failed to block AMPH‐mediated down‐regulation of WT‐hNET, NET‐specific blocker desipramine completely prevented AMPH‐induced down‐regulation. Furthermore, AMPH treatment had no effect on phospho‐CaMKII immunoreactivity. The inhibitory potency of AMPH was highest on hNET‐DM, intermediary on T258A and S259A single mutants and lowest on WT‐hNET. Single mutants exhibited partial resistance to AMPH‐mediated down‐regulation. AMPH accumulation was similar in cells expressing WT‐hNET or hNET‐DM. The results demonstrate that reduced plasma membrane insertion and enhanced endocytosis account for AMPH‐mediated NET down‐regulation, and provide the first evidence that T258/S259 motif is involved only in AMPH‐induced NET endocytosis that is desipramine‐sensitive, but PKC and CaMKII independent.