Francisco Javier Alvarez-Leefmans (Committee Chair), Kathrin Engisch (Committee Member), Dan Halm (Committee Member), James Olson (Committee Member), Michael Raymer (Committee Member)
Doctor of Philosophy (PhD)
Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF) and regulate its electrolyte composition. CPECs express both the Na+/ K+ ATPase and the Na+/ K+/ 2Cl- cotransporter 1 (NKCC1) on their apical membrane (CSF facing), deviating from the typical basolateral membrane location in chloride secretory epithelia. Given this unusual location of NKCC1 and the unknown intracellular Na+ and Cl- concentrations of CPECs, the cotransporter function in these cells is not understood. Further, the direction of net ion and associated water fluxes mediated by NKCC1 under basal physiological conditions in CPECs is controversial. Determining the direction of NKCC1- mediated fluxes is critical to understanding the function of this cotransporter in CPECs. If NKCC1 works in the net efflux mode, it may be involved in CSF secretion as suggested by some investigators. Conversely, if NKCC1 works in the net influx mode, as it does in the basolateral membrane of other chloride secreting epithelia, then it would have an absorptive function in CPECs and could be involved in CSF K+ regulation, as we and others propose. NKCC1-mediated ion and water fluxes are tightly linked; thus, their direction is inferred by measuring cell water volume (CWV) changes in calcein-loaded CPECs following NKCC1 inactivation. Both genetic and pharmacological inactivation of NKCC1 produces CPEC shrinkage of ~16 %, due to unbalanced solute and water effluxes. [Na+]i and [Cl-]i measurements using the fluorescent indicator dyes ANG-2 and MQAE show that [Na+]i = 8.4 +/- 1.0 mM and [Cl-]i = 62.1 +/- 3.5 mM. NKCC1 -/- CPECs have ~ 50% less [Cl-]i than WT. WT CPECs are exquisitely sensitive to isosmotic changes in [K+]o (+/- 2 mM), as shown by CWV responses. CPECs of NKCC1 -/- mouse lose the response to [K+]o. These results suggest that under basal conditions, NKCC1 is constitutively active and works in the inward mode near its thermodynamic equilibrium in CPECS. We propose NKCC1 has absorptive functions and contributes to the maintenance of normal and constant CWV. The Km for [K+]o and the thermodynamic forces driving NKCC1 transport in CPECs suggest that it functions as a sensor and regulator of CSF [K+].
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