MECHANISM OF CELLULAR REGULATION OF BICARBONATE TRANSPORTER FUNCTION : AN APPROACH WITH RUMINANT PAROTID TOWARD ITS UNDERSTANDING

虽然电致Na^+- HCO_<3^->共转运体（NBCe）被认为通过介导各种上皮内底侧HCO_<3^->内流或外流途径，在载体HCO_<3^->运输中起着至关重要的作用，但其在天然条件下的运输特性和细胞调控尚未得到很好的了解。本项目的主要目的是鉴定和表征从牛腮腺急性分离的腺泡细胞（BPA细胞）中的天然NBCe电流，已知该细胞产生大量富含HCO_<3^-> -的液体。利用全细胞膜片钳技术，我们首次证明了BPA细胞确实表达膜电流，这依赖于细胞外Na^+和HCO_<3^->的存在，被细胞外DIDS阻断，并且具有1 Na^+和2 HCO_<3^->的明显耦合比。利用分子生物学技术，我们还可以证明BPA细胞表达NBCe1-B的转录本，并且天然共转运体电流的电生理和药理学性质与HEK293细胞中表达的NBCe1-B重组电流相似。此外，我们可以提供证据表明，共转运蛋白活性可能有助于BPA细胞的静息膜电位。这些结果不仅首次提供了反刍动物腮腺中天然NBCe电流的电生理特征，而且为了解天然HCO_<3^->运输上皮中NBCe的细胞调控机制迈出了第一步。

Although the electrogenic Na^+- HCO_<3^-> cotransporter (NBCe) is believed to play a crucial role in vectorial HCO_<3^-> transport by mediating a basolateral HCO_<3^-> influx or efflux pathway in various epithelia, its transport properties and cellular regulation under native conditions have not been well understood. The main aim of the present project was to identify and characterize native NBCe currents in acinar cells (BPA cells) acutely isolated from bovine parotid, which is known to produce an enormous amount of HCO_<3^-> - rich fluid. Using the whole-cell patch-clamp technique, we could first demonstrate that BPA cells indeed express a membrane current, which is dependent on the presence of both extracellular Na^+ and HCO_<3^->, is blocked by extracellular DIDS, and has an apparent coupling ratio of 1 Na^+ to 2 HCO_<3^->. Using molecular biological techniques, we could also show that BPA cells express transcripts of NBCe1-B, and that electrophysiological and pharmacological properties of the native cotransporter current are similar to those of recombinant current of NBCe1-B expressed in HEK293 cells. Futhermore, we could provide evidence that the cotransporter activity likely contributes to the resting membrane potential of BPA cells. The results together not only provide the first electrophysiological characterization of native NBCe currents in a ruminant parotid, but also form a first step toward understanding cellular regulation mechanism of the NBCe in a native HCO_<3^-> transporting epithelium.