ELECTRONEUTRAL AND SLOW-RATE TRANSPORTERS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 电中性和慢速率转运蛋白的研究已经落后于产电转运蛋白的研究，由于难以监测转运速率。 电压钳用于测量与具有高转运速率的高密度生电转运蛋白的系统中的转运速率成比例的电流。 这是用于监测转运动力学的实时测定。 然而，电中性转运蛋白根本不产生电流，而慢速率转运蛋白产生的电流是不可检测的，除非转运蛋白密度达到高速率产电转运蛋白所用的1-10千倍。 我们已经发现，通过使用细胞外电化学传感器来监测在运输期间建立的梯度，存在用于监测运输的灵敏的实时测定。 通过监测浓度梯度中的两个点，我们可以计算与传输速率成比例的分析物的通量。 我们正在以这种方式探索许多不同的传输器。 我们目前正在调查的化学计量的非胃H+/K+，以帮助解决其他转运蛋白的研究。非胃H+/K+泵属于P型超家族，与Na+/K+泵和胃H+/K+泵的同源性均为65%。非胃H+/K+泵与胃H+/K+泵一样是一种电中性泵。然而，它似乎比H+泵出多10倍的Na+，使其更像Na+/K+泵。Na+或H+的化学计量从未与K+完全匹配。我们正在使用人非胃H+/K+泵（ATP 12 a）的异源表达与Na+/K+（ATP 1b）和胃H+/K+（ATP 4 b）的人β亚基的共表达，因为非胃H+/K+泵似乎不存在已知的β亚基。我们已经在非洲爪蟾卵母细胞中表达了这些亚基。然而，由于大量的内源性转运蛋白，我们一直不鼓励与此表达系统向前迈进。作为替代方案，我们将ATP 12 a和ATP 1b置于具有可表达的荧光指示剂的不同哺乳动物表达载体中以选择转染的细胞。与未转染的对照组相比，用ATP 12 a/ATP 1b瞬时转染的中国人卵巢（CHO）细胞显示出1000倍的H+外排（n=7）。为了进一步表征转运体的化学计量，我们使用具有离子选择性微电极的自参考的全细胞电压钳配置来表征所有3种离子的通量。全细胞电压钳配置使我们能够控制H+和Na+的胞质浓度。我们正在构建多离子选择性微电极，以便同时测量2种或更多种不同的离子，从而减少测量之间的差异，同时使用1个离子选择性微电极。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The study of electroneutral and slow rate transporters has lagged behind the study of electrogenic transporters due to the difficulty in monitoring transport rates. Voltage clamp is used to measure currents that are proportional to transport rates in systems with a high density of electrogenic transporters that have high transport rates. This is a real-time assay for monitoring transport kinetics. Electroneutral transporters, however, do not generate current at all while slow-rate transporters generate current that is not detectable unless transporter density reaches 1-10 thousand times greater than that used with high rate electrogenic transporters. We have found that a sensitive, real-time assay for monitoring transport exists by using extracellular, electrochemical sensors to monitor gradients established during transport. By monitoring two points in the concentration gradient we can calculate flux of the analyte that is proportional to transport rate. We are exploring a number of different transporters in this manner. We are currently investigating the stoichiometry of the nongastric H+/K+ in order to help troubleshoot the study of other transporters. The nongastric H+/K+ pump, is of the P-type superfamily with 65% homology to both the Na+/K+ pump and the gastric H+/K+ pump. The nongastric H+/K+ pump is an electroneutral pump like the gastric H+/K+ pump. However, it appears to pump 10 times more Na+ out than H+ making it more like the Na+/K+ pump. The stoichiometry for either Na+ or H+ has never fully been matched with K+. We are using heterologous expression of the human nongastric H+/K+ pump (ATP12a) with coexpression of the human beta subunits of the Na+/K+ (ATP1b) and gastric H+/K+ (ATP4b) as no known beta subunit appears to exist for the nongastric H+/K+ pump. We have expressed these subunits in Xenopus oocytes. However, due to the large number of endogenous transporters we have been discouraged from moving forward with this expression system. As an alternative we have placed ATP12a and ATP1b in different mammalian expression vectors with expressible fluorescent indicators to select transfected cells. Chinese Hamster Ovary (CHO) cells that have been transiently transfected with ATP12a/ATP1b show a 1000 fold greater efflux of H+ compared to untransfected controls (n=7). In order to characterize the stoichiometry of the transporter further we are using the whole cell voltage clamp configuration with self-referencing of ion-selective microelectrodes to characterize fluxes of all 3 ions. The whole cell voltage clamp configuration enables us to control the cytosolic concentration of H+ and Na+. We are in the process of building multi-ion-selective microelectrodes in order to perform measurement of 2 or more different ions at the same time in order to reduce variation between measurements while using 1 ion-selective microelectrode at a time.