The goal of this project is to express, characterize and, possibly, isolate the membrane protein(s) which form the amiloride-blockade Na channel of tight epithelial tissue. The amiloride-blockable Na channel is a major component of normal regulation of total body Na homeostasis; it represents the primary locus for control of discretionary Na resorption in the kidney. The properties of this channel appear to underlie specific features of the pathophysiology of electrolyte and fluid imbalance. The amiloride-blockable Na channel is found only in tissues which are highly specialized for Na reabsorption and the channel does not appear to be related to the Na channels of excitable tissues. The molecular basis and mechanism for the regulation of this Na channel in any cell is poorly understood; therefore, the Xenopus oocyte, which lacks any amiloride-blockable conductance, will be used as an mRNA expression system which, when injected with exogenous mRNA from tissues which normally contain the Na channels, will readily allow detection of the characteristic electrophysiological signal from amiloride- blockable Na channels. In this fashion, regulation of the amiloride-blockable Na channel can be studied in a controlled, simplified environment. But, in addition, the injected oocyte can also serve as a sensitive bioassay for Na channel-encoding mRNA, and therefore can allow the isolation of the cognate cDNA through standard molecular biological techniques. Because of the simplicity of the oocyte system, a characterization of the regulatory mechanisms controlling the channel will be much simpler than in any of the tissues in which it is normally found. In addition, it will be possible to prepare a cDNA library from this active mRNA pool. This library can be screened, using the oocyte to allow an identification of clones in the library which are cognates of all mRNA species essential for Na channel production. This approach to the isolation of the Na channels gene(s) is an alternative to more traditional methods of protein isolation which have so far been used in an effort to isolate and characterize the Na channel. The immediate advantage of the oocyte expression system is that a every step of the characterization and isolation process, the assay involves a functional channel. Such an approach will allow the development of a detailed molecular anatomy of this important channel, and further extend understanding of its role in physiological and pathophysiological processes.
| Hinton, C F; Eaton, D C (1989) Expression of amiloride-blockable sodium channels in Xenopus oocytes. Am J Physiol 257:C825-9 |
| Marunaka, Y (1988) Effects of internal Na and external K concentrations on Na/K coupling of Na,K-pump in frog skeletal muscle. J Membr Biol 101:19-31 |
