Localization of a Na⁺-K⁺-2Cl^- cotransporter in the rabbit lens

Earlier work from this laboratory demonstrated a bumetanide-inhibitable K⁺ uptake activity in cultured bovine lens epithelial cells, but not at the anterior surfaces of intact bovine lenses isolated in an Ussing-type chamber. Presently the distribution of the bumetanide-sensitive Na⁺-K⁺-2Cl^- cotransporter within the lens was re-examined. To complement previous results, ⁸⁶Rb⁺ uptake experiments were done in a chamber design that limited exposure of the radiolabel to specific surfaces of rabbit lenses under short-circuit conditions. In addition, the cotransporter protein (NKCC1. but not NKCC2) was immune-detected in Western blots. For the latter, membrane preparations were obtained from capsule-plus-epithelial specimens, and from three cortical fractions, i.e. the anterior, equatorial, and posterior regions, as well as a fifth, nuclear fraction. K⁺ influxes across the anterior-polar, equatorial, and posterior-polar surfaces were 0.375, 0.348 and 0.056 μEq (hr cm²)^-1 respectively, rates that were not significantly reduced by the presence of 0.1 mM bumetanide (P > 0.15, as unpaired data). In contrast, bumetanide-sensitive K⁺ influx rates were measured across the anterior and equatorial surfaces under hypertonic, but not under hypo-osmotic conditions. In culture, bumetanide and ouabain were equipotent in reducing by approximately half the K⁺ uptake of quiescent, rabbit lens epithelial cells under control, iso-osmotic conditions, indicating a cell-culture induced up-regulation of the cotransport activity by an undetermined mechanism. The immunoblotting of lens membrane proteins elicited approximately 170-180 kDa bands accordant with the identity of the NKCC1 isoform in the epithelial and cortical equatorial fractions. Thus, NKCC1 was readily demonstrated using membrane specimens taken from within the lens. Its activity in the intact organ may be activated by conditions fostering cell shrinkage, and perhaps, agents stimulating epithelial cell elongation, given its distribution within the lens.