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This Article Full Text Full Text (PDF) All Versions of this Article: 100/1/169    most recent 01007.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Rocha-González, H. I. Articles by Alvarez-Leefmans, F. J. Search for Related Content PubMed PubMed Citation Articles by Rocha-González, H. I. Articles by Alvarez-Leefmans, F. J.

Na⁺,K⁺,2Cl^– Cotransport and Intracellular Chloride Regulation in Rat Primary Sensory Neurons: Thermodynamic and Kinetic Aspects

Department of Pharmacology and Toxicology, Wright State University, Boonshoft School of Medicine, Dayton, Ohio

Submitted 10 September 2007; accepted in final form 2 April 2008

Adult primary afferent neurons are depolarized by GABA throughout their entire surface, including their somata located in dorsal root ganglia (DRG). Primary afferent depolarization (PAD) mediated by GABA released from spinal interneurons determines presynaptic inhibition, a key mechanism in somatosensory processing. The depolarization is due to Cl^– efflux through GABA_A channels; the outward Cl^– gradient is generated by a Na⁺,K⁺,2Cl^– cotransporter (NKCC) as first established in amphibians. Using fluorescence imaging microscopy we measured [Cl^–]_i and cell water volume (CWV) in dissociated rat DRG cells (P0–P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and calcein, respectively. Basal [Cl^–]_i was 44.2 ± 1.2 mM (mean ± SE), Cl^– equilibrium potential (E_Cl) was –27.0 ± 0.7 mV (n = 75). This [Cl^–]_i is about four times higher than electrochemical equilibrium. On isosmotic removal of external Cl^–, cells lost Cl^– and shrank. On returning to control solution, cells reaccumulated Cl^– and recovered CWV. Cl^– reaccumulation had Na⁺-dependent (SDC) and Na⁺-independent (SIC) components. The SIC stabilized at [Cl^–]_i = 13.2 ± 1.2 mM, suggesting that it was passive (E_Cl = –60.5 ± 3 mV). Bumetanide blocked CWV recovery and most (65%) of the SDC (IC₅₀ = 5.7 µM), indicating that both were mediated by NKCC. Active Cl^– uptake fell with increasing [Cl^–]_i and became negligible when [Cl^–]_i reached basal levels. The kinetics of active Cl^– uptake suggests a negative feedback system in which intracellular Cl^–regulates its own influx thereby keeping [Cl^–]_i constant, above electrochemical equilibrium but below the value that would attain if NKCC reached thermodynamic equilibrium.