Biophysical studies of human choline transporters linked to cholinergic synapses

描述(由申请人提供)：这项提议在胆碱能突触的研究中开辟了新的领域。乙酰胆碱(ACh)在突触间隙中被分解，其重新合成的一个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蛋白(HCHT)，以期了解它不仅在质膜中的作用，而且在将CHT运输到质膜的含有ACh的突触小泡(SVS)中也是如此。ACh是人类神经系统中的一种主要神经递质，调节注意力、奖赏、觉醒、睡眠、学习和记忆等认知过程。ACh不仅在骨骼肌，而且在平滑肌和心肌中对肌肉控制也是必不可少的。胆碱能神经传递缺陷导致阿尔茨海默病和精神分裂症，胆碱能信号减弱与神经肌肉疾病有关，如肌无力和迟发性运动障碍。因此，了解胆碱能传递的关键因素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是ACh持续合成的速率限制，但90%的CHTs是在含有ACh的SVS上，它在刺激时将CHTs输送到质膜。我们提出了CHT的生物物理分析，与其在质膜和SVS中的功能有关。由于CHTs可以运输ACh，因此一个悬而未决的问题是ACh SVS中的CHT活性。我们的方法包括在非洲爪哇卵母细胞中表达hCHT，以使用双电极电压钳(TEVC)来测量胆碱摄取和胆碱诱导电流。我们到目前为止的数据表明，胆碱摄取和诱导电流是pH敏感的(pKa为7.4)，在囊泡pH(5.5)时，胆碱摄取和电流被取消。此外，CHT特异性的半羟基-3结合表明胆碱结合部位附近的特定残基的H+滴定。这些观察结果使我们提出假设，质子使ACh-SVS上的CHTs失活，这使得它们能够在那里(用于输送)，同时防止ACh和H+从SV泄漏。我们将通过研究不同pH下ACh在CHTs中的传输来验证这一假设。我们还将在hCHT中寻找对pH敏感的残基(S)，并利用TEVC来研究摄取和电流的pH调节机制。最后，我们将描述hCHT，I89V的多态变异，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，它为我们大脑中的认知过程提供信息，如注意力、奖励、唤醒、睡眠、学习和记忆，它也是神经肌肉控制的关键，包括我们的手臂/腿运动和心跳。在乙酰胆碱被释放后，为了使正常的信号再次出现，胆碱必须被重新带入神经。这是由胆碱转运体完成的，其不寻常的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。PHS 398/2590(09/04版，2006年4月4日重新发布)页面续格式页面

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