Immunolocalization of the Na+–K+–2Cl- Cotransporter in Peripheral Nervous Tissue of Vertebrates

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Efflux of Cl through GABAA-gated anion channels depolarizes the cell bodies and intraspinal terminals of sensory neurons, and contributes to the generation of presynaptic inhibition in the spinal cord. Active accumulation of Cl inside sensory neurons occurs through an Na+–K+–2Cl cotransport system that generates and maintains the electrochemical gradient for this outward Cl current. We studied the immunolocalization of the Na+–K+–2Cl cotransporter protein using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the molecule. Western blots of frog, rat and cat dorsal root ganglion membranes revealed a single band of cotransporter immunoreactivity at ∼160 kDa, consistent with the molecular mass of the glycosylated protein. Deglycosylation with N-glycosidase F reduced the molecular mass to ∼135 kDa, in agreement with the size of the core polypeptide. Indirect immunofluorescence revealed strong cotransporter immunoreactivity in all types of dorsal root ganglion cell bodies in frog, rat and cat. The subcellular distribution of cotransporter immunoreactivity was different amongst species. Membrane labeling was more apparent in frog and rat dorsal root ganglion cell bodies than in cat. In contrast, cytoplasmic labeling was intense in cat and weak in frog, being intermediate in the rat. Cotransporter immunoreactivity also occurred in satellite cells, particularly in rat and cat dorsal root ganglia. The membrane region and axoplasm of sensory fibers were heavily labeled in cat and rat and less in frog. Three-dimensional reconstruction of confocal optical sections and dual immunolocalization with S-100 protein showed that the cotransporter immunoreactivity was prominently expressed in the nodal and paranodal regions of the Schwann cells. Ultrastructural immunolocalization confirmed the presence of immunoreactivity on the membranes of the axon and the Schwan cell in both the nodal region and the paranode. Treatment with sodium dodecylsulfate and β-mercaptoethanol also uncovered intense cotransporter immunoreactivity in Schmidt–Lanterman incisures at the light microscopic level. The localization of the Na+–K+–2Clcotransporter protein is consistent with its function as a Cl-accumulating mechanism in sensory neurons. Its distinctive presence in Schwann cells suggests that it could also be involved in K+ uptake from the extracellular space, particularly in the paranodal region of myelinated axons, thereby regulating the extracellular ionic environment and the excitability of axons.



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