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Article
Na+,K+,2Cl− Cotransport and Intracellular Chloride Regulation in Rat Primary Sensory Neurons: Thermodynamic and Kinetic Aspects
Journal of Neurophysiology
  • Héctor I. Rocha-González
  • Shihong Mao
  • Francisco J. Alvarez-Leefmans, Wright State University - Main Campus
Document Type
Article
Publication Date
7-1-2008
Abstract

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 GABAA 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 (ECl) 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 (ECl = −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.

DOI
10.​1152/​jn.​01007.​2007
Citation Information
Héctor I. Rocha-González, Shihong Mao and Francisco J. Alvarez-Leefmans. "Na+,K+,2Cl− Cotransport and Intracellular Chloride Regulation in Rat Primary Sensory Neurons: Thermodynamic and Kinetic Aspects" Journal of Neurophysiology Vol. 100 Iss. 1 (2008) p. 169 - 184 ISSN: 0022-3077
Available at: http://works.bepress.com/francisco_alvarez-leefmans/16/