Immunolocalization of the Na+-K+-2Cl(-) cotransporter in peripheral nervous tissue of vertebrates

Efflux of Cl- through GABA(A)-gated anion channels depolarizes the cell bod ies and intraspinal terminals of sensory neurons, and contributes to the ge neration of presynaptic inhibition in the spinal cord. Active accumulation of Cl- inside sensory neurons occurs through an Na+-K+-2Cl(-) cotransport s ystem that generates and maintains the electrochemical gradient for this ou tward Cl- current. We studied the immunolocalization of the Na+-K+-2Cl(-) c otransporter protein using a monoclonal antibody (T4) against a conserved e pitope in the C-terminus of the molecule. Western blots of frog, rat and ca t dorsal root ganglion membranes revealed a single band of cotransporter im munoreactivity at similar to 160 kDa, consistent with the molecular mass of the glycosylated protein. Deglycosylation with N-glycosidase F reduced the molecular mass to similar to 135 kDa, in agreement with the size of the co re polypeptide. Indirect immunofluorescence revealed strong cotransporter i mmunoreactivity in all types of dorsal root ganglion cell bodies in frog, r at 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, cytoplas mic labeling was intense in cat and weak in frog, being intermediate in the rat. Cotransporter immunoreactivity also occurred in satellite cells, part icularly in rat and cat dorsal root ganglia. The membrane region and axopla sm of sensory fibers were heavily labeled in cat and rat and less in frog. Three-dimensional reconstruction of confocal optical sections and dual immu nolocalization with S-100 protein showed that the cotransporter immunoreact ivity was prominently expressed in the nodal and paranodal regions of the S chwann cells. Ultrastructural immunolocalization confirmed the presence of immunoreactivity on the membranes of the axon and the Schwan cell in both t he nodal region and the paranode. Treatment with sodium dodecylsulfate and beta -mercaptoethanol also uncovered intense cotransporter immunoreactivity in Schmidt-Lanterman incisures at the light microscopic level. The localiz ation of the Na+-K+-2Cl(-) cotransporter protein is consistent with its fun ction as a Cl--accumulating mechanism in sensory neurons, Its distinctive p resence in Schwann cells suggests that it could also be involved in K+ upta ke from the extracellular space, particularly in the paranodal region of my elinated axone, thereby regulating the extracellular ionic environment and the excitability of axons. (C) 2001 IBRO. Published by Elsevier Science Ltd . All rights reserved.