Biophysical studies of human choline transporters linked to cholinergic synapses

描述（由申请人提供）：本提案为胆碱能突触的研究开辟了新的领域。乙酰胆碱（ACh）在突触间隙中被分解，其重新合成的必要条件是通过胆碱转运体（CHT）摄取胆碱。我们将在异源表达系统中研究人类胆碱转运体（hCHT），以期不仅了解其在质膜中的作用，而且了解将CHT运输到质膜的含乙酰胆碱突触囊泡（SVs）的作用。乙酰胆碱是人类神经系统中的一种主要神经递质，调节认知过程，如注意力、奖励、觉醒、睡眠、学习和记忆。乙酰胆碱不仅对骨骼肌，而且对平滑肌和心肌的肌肉控制也是必需的。胆碱能神经传递的缺陷与阿尔茨海默病和精神分裂症有关，胆碱能信号的减少与肌无力和迟发性运动障碍等神经肌肉疾病有关。因此，了解胆碱能传递的关键角色hCHT是一个具有广泛意义的重要问题。有趣的是，尽管突触前胆碱摄取限制了乙酰胆碱的持续合成，但90%的cht位于含乙酰胆碱的SVs上，后者在刺激时将cht传递到质膜。我们提出了一个生物物理分析相关的CHT在质膜和sv的功能。由于CHT可以转运乙酰胆碱，因此一个悬而未决的问题是ACh sv中CHT的活性。本研究采用双电极电压钳（TEVC）技术，在爪蟾卵母细胞中表达hCHT，测量胆碱摄取和胆碱诱导电流。到目前为止，我们的数据表明胆碱摄取和感应电流对pH值敏感（pKa为7.4），在囊泡pH值（5.5）下，胆碱摄取和感应电流被消除。此外，cht特异性的holhol3结合表明H+滴定了胆碱结合位点附近的特定残基。这些观察结果使我们得出这样的假设：质子使ACh- svs上的cht失活，这使得它们能够在那里（递送），同时防止ACh和H+从SV泄漏。我们将通过研究ACh在不同ph下通过CHTs的转运来验证这一假设。我们还将在hCHT中寻找ph敏感残基，并使用TEVC来研究摄取和电流的ph调节机制。最后，我们将描述hCHT的多态变异，I89V，它与人类焦虑增加和性欲降低相关。乙酰胆碱是一种神经递质，在我们的大脑中传递认知过程的信息，如注意力、奖励、觉醒、睡眠、学习和记忆，它对神经肌肉控制也很重要，包括我们的手臂/腿部运动和心跳。在乙酰胆碱被释放后，为了使正常的信号再次出现，胆碱必须被带回神经。这是由胆碱转运体完成的，其异常调节或基因突变与各种疾病有关，包括阿尔茨海默病、精神分裂症、舞蹈病和迟发性运动障碍。小灵通398/2590 （Rev. 09/04，重新发布4/2006）页延续格式页

DESCRIPTION (provided by applicant): This proposal opens new territory in the study of cholinergic synapses. Acetylcholine (ACh) is broken down in the synaptic cleft, and a requirement for its re-synthesis is choline uptake via the choline transporter (CHT). We will study the human choline transporter (hCHT) in a heterologous expression system with a view to understanding not only its role in the plasma membrane, but also in the ACh-containing synaptic vesicles (SVs) that traffic CHT to the plasma membrane. ACh is a major neurotransmitter in the human nervous systems that modulates cognitive processes such as attention, reward, arousal, sleep, learning, and memory. ACh is also essential for muscular control not only in skeletal muscle, but also in smooth muscle and cardiac muscle. Deficits in cholinergic neurotransmission contribute to Alzheimer's disease and schizophrenia, and diminished cholinergic signaling correlates with neuromuscular diseases, such as myasthenia and tardive dyskinetics. Thus, understanding a key player in cholinergic transmission, hCHT, is an important problem with broad implications. Interestingly, whereas presynaptic choline uptake is rate limiting for sustained ACh synthesis, 90% of CHTs are on ACh-containing SVs, which deliver CHTs to the plasma membrane upon stimulation. We propose a biophysical analysis of CHT relevant to its function on the plasma membrane and in SVs. Because CHTs can transport ACh, an open question is CHT activity in ACh SVs. Our methods include hCHT expression in Xenopus oocytes to measure choline uptake and choline-induced current using the two-electrode voltage-clamp (TEVC). Our data so far show that choline uptake and induced current are pH sensitive (pKa 7.4) and, at vesicular pH (5.5), choline uptake and current are abolished. Furthermore, CHT-specific hemicholinium-3 binding suggests H+ titration of a specific residue near the choline-binding site. These observations led us to the hypothesis that protons inactivate CHTs on ACh-SVs, which allows them to be there (for delivery) while at the same time preventing ACh and H+ leakage from the SV. We will test this hypothesis by investigating ACh transport through CHTs at various pH. We will also look for pH-sensitive residue(s) in hCHT, and use TEVC to study the pH-regulation mechanism of uptake and current. Finally, we will characterize the polymorphic variant in hCHT, I89V, which correlates with increased anxiety and reduced libido in humans. Acetylcholine is a neurotransmitter that carries messages for cognitive processes such as attention, reward, arousal, sleep, learning, and memory in our brain, and it is also is essential for neuromuscular control, including our arm/leg movements and heartbeat. After acetylcholine is released, for normal signaling to reoccur, choline must be taken up back into the nerve. This is accomplished by the choline transporter, whose unusual regulation or genetic mutations are related to various diseases including Alzheimer's disease, schizophrenia, choreics, and tardive dyskinetic. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page