Intracellular acidification in neurons induced by ammonium depends on KCC2 function

Abstract

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.

Intracellular ion homeostasis

“…Using calibration curves such as this, fluorescence values can be converted into absolute sodium concentrations mechanism responsible for both the acidifications and the large increases in sodium concentration observed in cultured astrocytes. Moreover, NKCC appears to be the main mechanism of inward NH 4 + transport in cultured astrocytes, findings which are in agreement with those published in 2006 by Titz et al [10], suggesting the involvement of NKCC in NH 4 + influx [10].…”

“…Therefore, NH 4 + influx in mature neurones probably occurs via alternative mechanisms. Published data [10] indicate the involvement of KCC in the NH 4 + influx into cultured neurones and supports the notion that NH 4 + influx pathways in neurones and astrocytes are different. However, the pathways for NH 4 + influx into neurones and astrocytes in situ are as yet unknown.…”

“…Cation-chloride cotransporters also contribute to NH 4 + influx, and the differential expression of these transporters between neurones and glial cells may contribute to the differences in sensitivity of the cell types to NH 4 + . In cultured neurones, NH 4 + uptake primarily involves potassium chloride cotransporters (KCC), whereas in cultured astrocytes the sodium-potassium-chloride cotransport (NKCC) may contribute [10].…”

Could tuning of the inhibitory tone involve graded changes in neuronal chloride transport?

“…Substitutions of designated amino acids with alanine were performed by sitedirected mutagenesis (QuikChange II, Agilent Technologies, USA) on an expression vector containing isoform 2 of the rat KCC2 DNA (Titz et al, 2006). Sequencing of the entire KCC2 protein-coding region confirmed the presence of the targeted substitution.…”

“…Therefore, in this study we used NH 4 þ -induced pH i shifts (Titz et al, 2006) to quantify transport exhibited by alanine dephosphorylation mimics, singly and in combination, in order to estimate the range of chloride extrusion possible by kinase/phosphatase regulation of KCC2 function. Additionally, we performed gramicidine perforated-patch recordings to measure the amplitude of GABA A currents in neurons expressing the different alanine constructs.…”