NKCC1 does not accumulate chloride in developing retinal neurons.
GABA excites immature neurons due to their relatively high intracellular chloride concentration. This initial high concentration is commonly attributed to the ubiquitous chloride cotransporter NKCC1, which uses a sodium gradient to accumulate chloride. Here we tested this hypothesis in immature retinal amacrine and ganglion cells. Western blotting detected NKCC1 at birth and its expression first increased, then decreased to the adult level. Immunocytochemistry confirmed this early expression of NKCC1 and localized it to all nuclear layers. In the ganglion cell layer, staining peaked at P4 and then decreased with age, becoming undetectable in adult. In comparison, KCC2, the chloride extruder, steadily increased with age localizing primarily to the synaptic layers. For functional tests, we used calcium imaging with fura-2 and chloride imaging with 6-methoxy-N-ethylquinolinium iodide. If NKCC1 accumulates chloride in ganglion and amacrine cells, deleting or blocking it should abolish the GABA-evoked calcium rise. However, at P0-5 GABA consistently evoked a calcium rise that was not abolished in the NKCC1-null retinas, nor by applying high concentrations of bumetanide (NKCC blocker) for long periods. Furthermore, intracellular chloride concentration in amacrine and ganglion cells of the NKCC1-null retinas was approximately 30 mM, same as in wild type at this age. This concentration was not lowered by applying bumetanide or by decreasing extracellular sodium concentration. Costaining for NKCC1 and cellular markers suggested that at P3, NKCC1 is restricted to Müller cells. We conclude that NKCC1 does not serve to accumulate chloride in immature retinal neurons, but it may enable Müller cells to buffer extracellular chloride.
Zhang
Ling-Li
LL
Dept of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104-6058, USA.
Delpire
Eric
E
Vardi
Noga
N
D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D004347:Drug Interactions; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D012160:Retina / Q000166:cytology* / Q000254:growth & development*; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000172:deficiency / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D002712:Chlorides; D009419:Nerve Tissue Proteins; C578878:Slc12a2 protein, mouse; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide; D005680:gamma-Aminobutyric Acid; D002118:Calcium