title: Differential contribution of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons.
abstract: 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.
authors.affiliation: Department of Molecular and Cell Biology, Katholieke Universiteit Leuven, Leuven, Belgium. jo.nsimy@gmail.com
authors.forename: J N
authors.initials: JN
authors.lastname: Chabwine
authors.forename: K
authors.initials: K
authors.lastname: Talavera
authors.forename: L
authors.initials: L
authors.lastname: Verbert
authors.forename: J
authors.initials: J
authors.lastname: Eggermont
authors.forename: J-M
authors.initials: JM
authors.lastname: Vanderwinden
authors.forename: H
authors.initials: H
authors.lastname: De Smedt
authors.forename: L
authors.initials: L
authors.lastname: Van Den Bosch
authors.forename: W
authors.initials: W
authors.lastname: Robberecht
authors.forename: G
authors.initials: G
authors.lastname: Callewaert
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D002034:Bumetanide / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004594:Electrophysiology; D004622:Embryo, Mammalian; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D006096:Gramicidin / Q000378:metabolism / Q000494:pharmacology; D007150:Immunohistochemistry; D007700:Kinetics; D015233:Models, Statistical; D009046:Motor Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D011919:Rats, Inbred Strains; D018079:Receptors, GABA / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism* / Q000502:physiology; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D002712:Chlorides; D018079:Receptors, GABA; D011963:Receptors, GABA-A; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D006096:Gramicidin; D005680:gamma-Aminobutyric Acid