Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice.
We have recently disrupted Slc12a2, the gene encoding the secretory Na-K-2Cl cotransporter in mice (NKCC1) (Delpire et al., 1999). Gramicidin perforated-patch and whole-cell recordings were performed to study GABA-induced currents in dorsal root ganglion (DRG) neurons isolated from wild-type and homozygote NKCC1 knock-out mice. In wild-type DRG neurons, strong GABA-evoked inward current was observed at the resting membrane potential, suggesting active accumulation of Cl(-) in these cells. This GABA-induced current was blocked by picrotoxin, a GABA(A) receptor blocker. The strong Cl(-) accumulation that gives rise to depolarizing GABA responses is caused by Na-K-2Cl cotransport because reduction of external Cl(-) or application of bumetanide induced a decrease in [Cl(-)](i), whereas an increase in external K(+) caused an apparent [Cl(-)](i) accumulation. In contrast to control neurons, little or no net current was observed at the resting membrane potential in homozygote NKCC1 mutant DRG neurons. E(GABA) was significantly more negative, demonstrating the absence of Cl(-) accumulation in these cells. Application of bumetanide induced a positive shift of E(GABA), suggesting the presence of an outward Cl(-) transport mechanism. In agreement with an absence of GABA depolarization in DRG neurons, behavioral analysis revealed significant alterations in locomotion and pain perception in the knock-out mouse. Our results clearly demonstrate that the Na-K-2Cl cotransporter is responsible for [Cl(-)](i) accumulation in DRG neurons and that via regulation of intracellular Cl(-), the Na-K-2Cl cotransporter participates in the modulation of GABA neurotransmission and sensory perception.
Sung
K W
KW
Departments of Anesthesiology, Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennesse, USA.
Kirby
M
M
McDonald
M P
MP
Lovinger
D M
DM
Delpire
E
E
D000818:Animals; D001522:Behavior, Animal; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D000075202:Contraindications; D005260:Female; D018756:GABA Antagonists / Q000494:pharmacology; D058787:GABA-A Receptor Antagonists; D020233:Gait Disorders, Neurologic / Q000235:genetics; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006096:Gramicidin / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010147:Pain Measurement; D018408:Patch-Clamp Techniques; D010852:Picrotoxin / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
D002034:Bumetanide; D002352:Carrier Proteins; D002712:Chlorides; D018756:GABA Antagonists; D058787:GABA-A Receptor Antagonists; D006096:Gramicidin; D010852:Picrotoxin; D011188:Potassium; D011963:Receptors, GABA-A; D028021:Sodium-Potassium-Chloride Symporters; D005680:gamma-Aminobutyric Acid