NEUROTRANSMITTER TRANSPORTER STRUCTURE/FUNCTION

本实验室一直在研究半胱氨酸取代和修饰对5-羟色胺转运体(SERT)的影响。我们将通过检查转运蛋白的两个区域来继续这些努力，其中建模工作表明以前被鉴定为属于跨膜结构域的序列可能位于外部或内部环中。在含有外环1(EL1)和跨膜结构域2(TM2)的区域，我们将检验TM2以β构型跨膜的预测，因此EL1比预测的要大。通过确定反应性和活性修改的模式与EL1和TM2之间的转换最一致的地方，我们希望区分SERT拓扑的原始模型和当前的预测。我们已经证明，SERT的内部半胱氨酸残基与MTSEA-生物素反应，细胞被洋地黄素渗透，即使没有外部半胱氨酸可用。我们建议利用这一性质通过取代预测位于内环中的七个半胱氨酸残基来确定内环残基的暴露。我们希望在完整的细胞和膜制剂中产生一种对MTSEA-生物素不起反应的SERT形式。在内环(IL)中替换原来的半胱氨酸(IL)，如IL5，可以增加SERT对MTSEA-生物素的反应性。我们将测试TM10和IL5区域的残基与细胞质的暴露，方法是每次用半胱氨酸取代一个残基，并测试洋地黄对MTSEA-生物素的依赖反应性和修饰的功能后果。该区域的拓扑建模存在相当大程度的不确定性。我们希望建立这种方法来确定内部环区的暴露程度，并将其应用于TM10-IL5区域的SERT拓扑。

This laboratory has been examining the effects of cysteine substitution and modification in the 5-HT transporter (SERT). We will continue these efforts by examining two regions of the transporter where modeling efforts suggest that sequence previously identified as belonging to transmembrane domains may be in external or internal loops. In the region containing external loop 1 (EL1) and transmembrane domain 2 (TM2), we will test the prediction that TM2 crosses the membrane in a beta configuration and EL1 is consequently larger than predicted. By determining where the patterns of reactivity and activity modification are most consistent with the transition between EL1 and TM2, we hope to distinguish between the original model of SERT topology and the current predictions. We have shown that internal cysteine residues of SERT react with MTSEA-biotin with cells are permeabilized with digitonin, even if non external cysteines are available. We propose to use this property to determine exposure of internal loop residues by substituting the seven-cysteine residues predicted to lie in internal loops. We expect to generate a form of SERT that is unreactive toward MTSEA-biotin both in intact cells and in membrane preparations. Replacing the original cysteine in internal loops (IL) such as IL5 should increase the reactivity of SERT toward MTSEA-biotin. We will test the exposure of residues in the region of TM10 and IL5 to the cytoplasm by replacing residues one at a time with cysteine, and testing for digitonin-dependent reactivity toward MTSEA-biotin and functional consequences of modification. A considerable degree of uncertainty exists in the topological modeling of this region. We hope to establish this method to determine exposure of internal loop domains and to apply to the topology of SERT in the TM10-IL5 region.