Involvement of threonine 258 and serine 259 motif in amphetamine-induced norepinephrine transporter endocytosis.
Involvement of threonine 258 and serine 259 motif in amphetamine-induced norepinephrine transporter endocytosis.
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DOI：
10.1111/j.1471-4159.2010.06898.x
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发表时间：
2010-10
影响因子：
4.7
通讯作者：
Jayanthi LD
中科院分区：
文献类型：
--
作者：
Annamalai B;Mannangatti P;Arapulisamy O;Ramamoorthy S;Jayanthi LD
D-Amphetamine (AMPH) downregulates the norepinephrine (NE) 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 (HTR) cells expressing the wild-type (WT)-hNET and the hNET double mutant (hNET-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. While PKC or calcium/calmodulin-dependent kinase-II (CaMKII) inhibition or depletion of calcium failed to block AMPH-mediated downregulation of WT-hNET, NET-specific blocker desipramine (DMI) completely prevented AMPH-induced downregulation. 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 downregulation. 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 downregulation, and provide the first evidence that T258/S259 motif is involved only in AMPH-induced NET endocytosis that is DMI-sensitive, but PKC and CaMKII independent.
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