Absence of Na+/K+/2Cl− cotransporter activity in corneal endothelial cell plasma membrane vesicles

Abstract

There are various models to describe coupling between transmembrane Na− flux and transendothelial net HCO−3 flux in the cornea, including a direct coupling via a Na+/HCO−3 cotransporter or an indirect coupling utilizing a Na+/K+/2Cl− cotransporter further coupled to a Cl−/HCO−3 exchanger. In this study we investigate the presence of Na+/K+/2Cl− cotransporter activity by using corneal endothelial cell plasma membrane vesicles. By controlling the external and internal compartments of the vesicles we were able to monitor Na+ movement across the membranes.If the Na+/K+/2Cl− cotransporter is present in the corneal endothelial cell membrane vesicles then the presence of K+ should lead to a significant increase in uptake of 22Na+. The rate of accumulation of 22Na+ in the presence of K+ was 25.7 ± 2.8 nmoles Na+ mg−1 protein 15 s−1 which was not significantly different from that in the absence of K+ (23.8 ± 2.0 nmoles Na+ mg−1 protein 15 s−1) indicating that Na+ uptake into the vesicles was not dependent on K+.The present work provides no evidence for Na+/K+/2Cl− cotransport in corneal endothelial cell plasma membrane vesicles. The nature of the coupling mechanism between HCO−3 and Na+ is still unresolved.