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Updated: May 23, 2026
Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting
Published on: December 9, 2022
Molecular and functional expression of cation-chloride cotransporters in dorsal root ganglion neurons during
Shihong Mao1, Tomás Garzon-Muvdi, Mauricio Di Fulvio
1Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435-0001, USA.
Abstract:
GABA depolarizes and excites central neurons during early development, becoming inhibitory and hyperpolarizing with maturation. This "developmental shift" occurs abruptly, reflecting a decrease in intracellular Cl(-) concentration ([Cl(-)](i)) and a hyperpolarizing shift in Cl(-) equilibrium potential due to upregulation of the K(+)-Cl(-) cotransporter KCC2b, a neuron-specific Cl(-) extruder. In contrast, primary afferent neurons (PANs) are depolarized by GABA throughout adulthood because of expression of NKCC1, a Na(+)-K(+)-2Cl(-) cotransporter that accumulates Cl(-) above equilibrium. The GABA(A)-mediated depolarization of PANs determines presynaptic inhibition in the spinal cord, a key mechanism gating somatosensory information. Little is known about developmental changes in Cl(-) transporter expression and Cl(-) homeostasis in PANs. Whether NKCC1 is expressed in PANs of all phenotypes or is restricted to subpopulations (e.g., nociceptors) is debatable. Likewise, whether PANs express KCC2s is controversial. We investigated NKCC1 and K(+)-Cl(-) cotransporter expression in rat and mouse dorsal root ganglion (DRG) neurons with molecular methods. Using fluorescence imaging microscopy, we measured [Cl(-)](i) in acutely dissociated rat DRG neurons (P0-P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and classified with phenotypic markers. DRG neurons of all sizes express two NKCC1 mRNAs, one full-length and a shorter splice variant lacking exon 21. Immunolabeling with validated antibodies revealed ubiquitous expression of NKCC1 in DRG neurons irrespective of postnatal age and phenotype. As maturation progresses [Cl(-)](i) decreases gradually, persisting above equilibrium in >95% mature neurons. DRG neurons express mRNAs for KCC1, KCC3s, and KCC4, but not for KCC2s. Mechanisms underlying PANs' developmental changes in Cl(-) homeostasis are discussed and compared with those of central neurons.
