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.
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