Genetic and pharmacological inactivation of apical Na⁺-K⁺-2Cl⁻ cotransporter 1 in choroid plexus epithelial cells reveals the physiological function of the cotransporter

Abstract

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF). They express Na+-K+-ATPase and Na+-K+-2Cl− cotransporter 1 (NKCC1) on their apical membrane, deviating from typical basolateral membrane location in secretory epithelia. Given this peculiarity, the direction of basal net ion fluxes mediated by NKCC1 in CPECs is controversial, and cotransporter function is unclear. Determining the direction of basal NKCC1-mediated fluxes is critical to understanding the function of apical NKCC1. If NKCC1 works in the net efflux mode, it may be directly involved in CSF secretion. Conversely, if NKCC1 works in the net influx mode, it would have an absorptive function, contributing to intracellular Cl− concentration ([Cl−]i) and cell water volume (CWV) maintenance needed for CSF secretion. We resolve this long-standing debate by electron microscopy (EM), live-cell-imaging microscopy (LCIM), and intracellular Na+ and Cl− measurements in single CPECs of NKCC1+/+ and NKCC1−/− mouse. NKCC1-mediated ion and associated water fluxes are tightly linked, thus their direction is inferred by measuring CWV changes. Genetic or pharmacological NKCC1 inactivation produces CPEC shrinkage. EM of NKCC1−/− CPECs in situ shows they are shrunken, forming large dilations of their basolateral extracellular spaces, yet remaining attached by tight junctions. Normarski LCIM shows in vitro CPECs from NKCC1−/− are ~17% smaller than NKCC1+/+. CWV measurements in calcein-loaded CPECs show that bumetanide (10 μM) produces ~16% decrease in CWV in NKCC1+/+ but not in NKCC1−/− CPECs. Our findings suggest that under basal conditions apical NKCC1 is continuously active and works in the net inward flux mode maintaining [Cl−]i and CWV needed for CSF secretion.

Novel Human NKCC1 Mutations Cause Defects in Goblet Cell Mucus Secretion and Chronic Inflammation

“…This phenotype was not unexpected because NKCC1 is involved in a regulatory volume increase after a hypertonic shock in many cells 26 and because shrinkage was observed in other tissues (eg, choroid plexus) in the absence of NKCC1 function. 27,28 The first major epithelial deficit found in these studies relates to mucus secretion. Goblet cells are the cells in the intestine that secrete gel-forming mucins.…”

Modulation of brain cation-Cl− cotransport via the SPAK kinase inhibitor ZT-1a

“…NKCC1 expressed in the apical CPe contributes approximately half of CSF production, though its mechanism remains unclear, in view of its unique apical localization in the CPe compared with its basolateral position in all other epithelia 1 . Indeed, some have proposed inward NKCC1 flux to be required for apical K + recycling and continued CSF production 86,87 , while others have provided evidence for net ion efflux with obligatory water transport directly contributing to CSF production 1,3 . Nonetheless, in a rat model of hemorrhagic hydrocephalus, intraventricular hemorrhage causes a Toll-like receptor 4 (TLR4)-and NF-κB-dependent inflammatory response in CPe associated with~3-fold increase in bumetanide-sensitive CSF secretion 3 .…”

Elusive role of the Na-K-2Cl cotransporter in the choroid plexus

“…However, two recent papers published in Nature Medicine (9) and Nature Communication (18) argued for unconventional outward NKCC1 transport in choroid plexus. A paper in this issue of American Journal of Physiology-Cell Physiology challenges this view and supports the conventional inward transport (5). Can both sides of the argument be right?…”

“…Direction of transport is inward below the diagonal line (dashed area) and outward above the line. The red dot represents the product ratio of Steffensen et al (18) and the blue dot represents the product ratio of Gregoriades et al (5). B: ranges of intracellular Cl Ϫ concentrations at different [Na ϩ ]CP needed to facilitate outward NKCC1-mediated flux.…”