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Differential contribution of the Na⁺-K⁺-2Cl^– cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons

J. N. Chabwine^*,1, K. Talavera^*, L. Verbert^*, J. Eggermont^*, J.-M. Vanderwinden, H. De Smedt^*, L. Van Den Bosch, W. Robberecht and G. Callewaert^*

^* Department of Molecular and Cell Biology and

Laboratory of Neurobiology, Katholieke Universiteit Leuven, Vesalius Research Center, VIB, Leuven, Belgium; and

Laboratory of Neurophysiology, Université Libre de Bruxelles, Brussels, Belgium

¹Correspondence: Department of Molecular Cell Biology, Campus Gasthuisberg O&N1, Herestraat, 49/802, B-3000 Leuven, Belgium. E-mail: jo.nsimy{at}gmail.com

Plasma membrane chloride (Cl^–) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl^– uptake mechanism) in Cl^– handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated patch-clamp recordings revealed that DRGNs accumulate intracellular Cl^– through a bumetanide- and Na⁺-sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of Cl^– during GABA_A receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting Cl^– concentration (i.e., [Cl^–]_i decrease) was similar in both cell types and was unaffected by lowering the extracellular Na⁺ concentration. In contrast, the recovery on depletion (i.e., [Cl^–]_i increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na⁺. The experimental observations could be reproduced by a mathematical model for intracellular Cl^– kinetics, in which DRGNs show higher NKCC1 activity and smaller Cl^–-handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [Cl^–]_i, whereas lower NKCC1 activity in MNs allows large [Cl^–]_i variations during neuronal activity.—Chabwine, J. N., Talavera, K., Verbert, L., Eggermont, J., Vanderwinden, J.-M., De Smedt, H., Van Den Bosch, L., Robberecht, W., Callewaert, G. Differential contribution of the Na⁺-K⁺-2Cl^– cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons.

Key Words: GABA receptor • bumetanide • primary afferent depolarization • kinetic model