CHARACTERIZATION OF THE STOICHIOMETRY OF THE NA+-BICARBONATE COTRANSPORTER

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。子项目及 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 研究中心，而研究中心不一定是研究者的研究机构。 离子选择性微电极和电压钳自参考Na+-碳酸氢盐协同转运蛋白的化学计量表征。由于最终碳酸氢盐浓度取决于培养基的pH值、温度、离子强度和周围CO2的分压，因此使用碳酸氢盐缓冲溶液进行操作是繁琐的。虽然碳酸氢盐可以计算，但计算实际上只是一个估计。早期的工作将Na+ /碳酸氢盐转运蛋白的化学计量置于1：2和1：3之间的某处。 后来证明，当转运蛋白在一种内源性细胞系中时，它显示1：2的化学计量，而当在另一种内源性细胞系中时，它显示1：3的化学计量。 我们的同事最近在异源表达系统中表达了转运蛋白，以确定化学计量并确定化学计量可以改变的机制。他们目前用于确定Na+碳酸氢盐化学计量的设置导致了差异。使用过表达Na+/碳酸氢盐转运蛋白的细胞，我们通过直接测量在电压钳下保持的细胞旁边的H+通量来确定转运蛋白的化学计量。通过测量穿过质膜的电流沿着细胞外H+通量，我们可以使用最佳拟合模型来确定转运蛋白的化学计量，甚至推测泵是否通过碳酸氢盐与碳酸盐，这是最近的争议。

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. Characterization of the stoichiometry of the Na+-bicarbonate cotransporter with self-referencing of ion-selective microelectrodes and voltage clamp. Working with bicarbonate buffered solutions is tedious due to the fact that the final bicarbonate concentration is based on the pH, temperature, ionic strength of the medium and the partial pressure of surrounding CO2. While bicarbonate can be calculated, the calculation is really only an estimate. Early work placed the stoichiometry of the Na+ /bicarbonate transporter at somewhere between 1:2 and 1:3. Later it was shown that when the transporter is in one endogenous cell line it shows a 1:2 stoichiometry and when in another endogenous cell line it shows a stoichiometry of 1:3. Our colleagues have recently expressed the transporter in a heterologous expression system to nail down the stoichiometry and determine the mechanism by which the stoichiometry can be changed. Their current setup for determining the stoichiometry of the Na+ bicarbonate has led to discrepancies. Using cells overexpressing the Na+/bicarbonate transporter, we are identifying the stoichiometry of the transporter by directly measuring H+ fluxes next to cells held under voltage clamp. By measuring the current passing through the plasma membrane along with the extracellular H+ flux we can use best fit models to determine the transporters stoichiometry and even speculate as to whether the pump is passing bicarbonate vs carbonate a more recent controversy.