Abstract
Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.
Highlights
serotonin transporter (SERT) is of particular interest in neurobiology because it is the molecular target of several drugs of abuse and many therapeutic agents used to treat psychiatric disorders
For determination of MTSEA or (2-sulfonatoethyl)methanethiosulfonate (MTSES) sensitivity, the membranes were incubated with MTSEA or MTSES at varying concentrations for 15 min at room temperature following the initial washing of the membranes, and the MTS reagent was removed by washing the membranes three times with binding buffer
The region is likely intracellular, because mutants with cysteine at these positions were inactivated by MTSEA in membrane preparations (Figs. 4, 5, and 8) but did not react with extracellular MTSEA in intact cells (Table II)
Summary
Mutagenesis—Mutant transporters were generated by site-directed mutagenesis using the QuikChangeTM kit (Stratagene). Cells were preincubated with 1 mM MTSEA for 10 min at room temperature in PBS/CM and washed three times with 100 l of PBS/CM to quench unreacted MTSEA, and the transport assay was performed as described above. Whole Cell Binding Assay—Cells expressing each of the IL2 mutants were preincubated with 1 mM MTSEA for 10 min at room temperature and washed three times with 100 l of PBS/CM. Binding was allowed to proceed for 1.5 h at room temperature with gentle rocking, and the assay was terminated by washing the cells three times with 100 l of ice-cold PBS. For determination of MTSEA or (2-sulfonatoethyl)methanethiosulfonate (MTSES) sensitivity, the membranes were incubated with MTSEA or MTSES at varying concentrations for 15 min at room temperature following the initial washing of the membranes, and the MTS reagent was removed by washing the membranes three times with binding buffer. Statistical analysis was performed using Student’s paired t tests
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