Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an alpha-Helical Conformation

@article{citeulike:2615721, 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 2{beta}-carbomethoxy-3{beta}-(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. 10.1074/jbc.M504087200}, author = {Zhang, Yuan-Wei and Rudnick, Gary }, citeulike-article-id = {2615721}, doi = {10.1074/jbc.M504087200}, journal = {J. Biol. Chem.}, keywords = {methods}, month = {September}, number = {35}, pages = {30807--30813}, posted-at = {2008-03-31 11:23:08}, priority = {2}, title = {Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an {alpha}-Helical Conformation}, url = {http://dx.doi.org/10.1074/jbc.M504087200}, volume = {280}, year = {2005} }

TY - JOUR ID - citeulike:2615721 L3 - citeulike-article-id:2615721 TI - Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an {alpha}-Helical Conformation JF - J. Biol. Chem. VL - 280 IS - 35 SP - 30807 EP - 30813 N2 - 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 2{beta}-carbomethoxy-3{beta}-(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. 10.1074/jbc.M504087200 KW - methods AU - Zhang, Yuan-Wei AU - Rudnick, Gary PY - 2005/09/02/ UR - http://dx.doi.org/10.1074/jbc.M504087200 ER -