Intracellular acidification in neurons induced by ammonium depends on KCC2 function.
The Cl(-)-extruding neuron-specific K(+)-Cl(-) cotransporter KCC2, which establishes hyperpolarizing inhibition, can transport NH(4) (+) instead of K(+). It is, however, not clear whether KCC2 provides the only pathway for neuronal NH(4) (+) uptake. We therefore investigated NH(4) (+) uptake in cultured rat brain neurons. In neurons cultured for > 4 weeks, the response to NH(4)Cl applications (5 mM) consisted of an alkaline shift which reversed to an acid shift within seconds. Rebound acid shifts which followed brief applications of NH(4)Cl were blocked by furosemide (100 microM). They were rather insensitive to bumetanide (1 and 100 microM), in contrast to those induced in cultured glial cells. Rebound acid shifts persisted in the presence of 1 mM Ba(2+) and in Na(+)-free solution but were inhibited by extracellular K(+). In neurons with depolarizing GABA responses, indicating the absence of functional KCC2, applications of NH(4)Cl barely induced an acidosis. However, large rebound acid shifts occurred in neurons that had changed their GABA response from Ca(2+) increases to Ca(2+) decreases. Rebound acid shifts continued to increase even after the change in the GABA response had occurred and could be induced earlier in neurons transfected with KCC2 cDNA. We conclude that KCC2 provides the main pathway for fast neuronal NH(4) (+) uptake. Therefore, NH(4)Cl-induced rebound acid shifts can be used to indicate the development of KCC2 function. Further, the well known up-regulation of KCC2 function during development has the inevitable consequence of opening a major pathway for NH(4) (+) influx, which can be relevant under pathophysiological conditions.
Titz
Stefan
S
Institut für Physiologie und Pathophysiologie und Interdisziplinäres Zentrum für Neurowissenschaften, Universität Heidelberg, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany.
Hormuzdi
Sheriar
S
Lewen
Andrea
A
Monyer
Hannah
H
Misgeld
Ulrich
U
D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002478:Cells, Cultured; D003952:Diagnostic Imaging / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian; D005260:Female; D005665:Furosemide / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects*; D007150:Immunohistochemistry / Q000379:methods; D042541:Intracellular Space / Q000502:physiology*; D008636:Mesencephalon / Q000166:cytology; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D011189:Potassium Chloride / Q000494:pharmacology; D011247:Pregnancy; D011758:Pyrroles / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D027981:Symporters / Q000502:physiology*; D013997:Time Factors; D014162:Transfection / Q000379:methods; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology; D000096922:K Cl- Cotransporters
D002034:Bumetanide; D002118:Calcium; D002120:Calcium Channel Agonists; D005665:Furosemide; D049452:Green Fluorescent Proteins; D011189:Potassium Chloride; D011758:Pyrroles; D000644:Quaternary Ammonium Compounds; D049994:Sodium Potassium Chloride Symporter Inhibitors; D027981:Symporters; D005680:gamma-Aminobutyric Acid; D000096922:K Cl- Cotransporters; C071658:FPL 64176