Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na+/K+/Cl-cotransport system and a K+channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na+/K+/Cl-cotransporter and K+channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of86Rb+uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two86Rb+fluxes. (A) A furosemide-inhibited86Rb+flux in the absence of Na+(K+-K+exchange). This flux is stimulated by an inward Na+gradient (Na+/K+cotransport) and is inhibited also by bumetanide. (B) A Ba2+-inhibited86Rb+flux, through the K+channel. Luminal membranes containing the Na+/K+/Cl-cotransporter and K+channels, and basolateral membranes containing the Na+/K+pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na+/K+/Cl-cotransporter and K+channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column. © 1985.
Burnham, C., Karlish, S. J. D., & Jørgensen, P. L. (1985). Identification and reconstitution of a Na+/K+/Cl-cotransporter and K+channel from luminal membranes of renal red outer medulla. BBA - Biomembranes, 821(3), 461–469. https://doi.org/10.1016/0005-2736(85)90051-3