Lech Kiedrowski

Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in Motor Learning and Memory

Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-exchangers belonging to both the K+-independent NCX family and the K+-dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca2+ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K+ -dependent Na+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.

Differential contribution of plasmalemmal Na+/Ca2+ exchange isoforms to sodium-dependent calcium influx and NMDA excitotoxicity in depolarized neurons

Inhibition of Na+,K+‐ATPase during NMDA applications greatly increased NMDA‐induced excitotoxicity in primary cultures of forebrain neurons (FNs), but not in cerebellar granule cells (CGCs). Because Na+,K+‐ATPase inhibition promotes reversal of plasmalemmal Na+/Ca2+ exchangers, we compared the activities of reversed K+‐independent (NCX) and K+‐dependent (NCKX) Na+/Ca2+ exchangers in these cultures. To this end, we measured gramicidin‐induced and Na+‐dependent elevation in cytosolic [Ca2+] ([Ca2+]c) that represents Ca2+ influx via reversed NCX and NCKX; NCX activity was dissected out by removing external K+. The [Ca2+]c elevations mediated by NCX alone, and NCX plus NCKX combined, were 17 and 6 times more rapid in FNs than in CGCs, respectively. Northern blot analysis showed that FNs preferentially express NCX1 whereas CGCs expressed NCX3. Differences in expression of other isoforms (NCX2, NCKX2, NCKX3 and NCKX4) were less pronounced. We tested whether the NCX or NCKX family of exchangers contributes most to the toxic NMDA‐induced Ca2+ influx in depolarized neurons. We found that in FNs, inhibition of NCX alone was sufficient to significantly limit NMDA excitotoxicity, whereas in CGCs, inhibition of both NCX and NCKX was required. The data suggest that the high activity of NCX isoforms expressed in FNs, possibly NCX1, sensitizes these neurons to NMDA excitotoxicity.

Instrumental role of Na+ in NMDA excitotoxicity in glucose‐deprived and depolarized cerebellar granule cells

In glucose‐deprived cerebellar granule cells, substitution of extracellular Na+ with Li+ or Cs+ prevented N‐methyl‐d‐aspartate (NMDA)‐induced excitotoxicity. NMDA stimulated 45Ca2+ accumulation and ATP depletion in a Na‐dependent manner, and caused neuronal death, even if applied while Na,K‐ATPase was inhibited by 1 mm ouabain. The cells treated with NMDA in the presence of ouabain accumulated sizable 45Ca2+ load but most of them failed to elevate cytosolic [Ca2+] upon mitochondrial depolarization. Na/Ca exchange inhibitor, KB‐R7943, inhibited Na‐dependent and NMDA‐induced 45Ca2+ accumulation but only if Na,K‐ATPase activity was compromised by ouabain. In cells energized by glucose and exposed to NMDA without ouabain, KB‐R7943 reduced NMDA‐elicited ionic currents by 19% but failed to inhibit 45Ca2+ accumulation. It appears that a large part of NMDA‐induced Ca2+ influx in depolarized and glucose‐deprived cells is mediated by reverse Na/Ca exchange. A high level of reverse Na/Ca exchange operation is maintained by a sustained Na+ influx via NMDA channels and depolarization of the plasma membrane. In cells energized by glucose, however, most Ca2+ enters directly via NMDA channels because Na,K‐ATPase regenerating Na+ and K+ concentration gradients prevents Na/Ca exchange reversal. Since under these conditions Na/Ca exchange extrudes Ca2+, its inhibition destabilizes Ca2+ homeostasis.

AMPA-mediated excitotoxicity in oligodendrocytes : role for Na+-K+-Cl- co-transport and reversal of Na+/Ca2+ exchanger

We investigated the role of Na+–K+–Cl− co‐transporter isoform 1 (NKCC1) and reversal of Na+/Ca2+ exchanger (NCXrev) in glutamate‐mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6–8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na+/Ca2+ exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1‐mediated Na+ influx. In the presence of a specific AMPA receptor inhibitor 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ‐induced sustained rise led to mitochondrial Ca2+ accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ‐elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCXrev. These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1‐mediated Na+ influx. The latter triggers NCXrev and NCXrev‐mediated overload and compromises mitochondrial function and cellular viability.

High activity of K+-dependent plasmalemmal Na+/Ca2+ exchangers in hippocampal CA1 neurons.

Ca2+ influx via reversed K+-dependent (NCKX) and/or K+-independent (NCX) plasmalemmal Na+/Ca2+ exchangers may play a role in neuronal death following global brain ischemia to which CA1 neurons are particularly vulnerable. Therefore, this work tested whether the rates of Ca2+ influx via reversed NCKX or NCX in cultured rat CA1 neurons differ from those in forebrain neurons (FNs) or cerebellar granule cells (CGCs). The NCKX-mediated Ca2+ influx was several times more rapid in CA1 neurons than in FNs or CGCs and was not affected by Na+/Ca2+ exchange inhibitors, KB-R7943 or bepridil. NCKX reversal inhibitors are not yet available. Their development would greatly facilitate further testing the role of NCKX in ischemic death of CA1 neurons.

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate - The role of pH and sodium

J. Neurochem. (2011) 117, 231–243.

Preferential expression of plasmalemmal K-dependent Na+/Ca2+ exchangers in neurons versus astrocytes.

Numerous isoforms of plasmalemmal K-dependent (NCKX) and K-independent (NCX) Na+/Ca2+ exchangers are expressed in the brain. The physiological functions of each isoform are presently unknown. Therefore, in this study, we compared expression of NCKX and NCX transcripts between primary cultures of cerebellar granule cells, and astrocytes. Northern blot analysis showed that granule cells expressed NCKX2, NCKX3, NCKX4 and NCX3, whereas astrocytes expressed primarily NCX1. Consistent with this molecular characterization, a significant fraction of 45Ca2+ accumulation in Na-loaded granule cells, but not in astrocytes, depended on external K+. This is the first demonstration of native NCKX activity in neurons derived from the central nervous system. Our data suggest that NCKX isoform expression may correspond to the unique Ca2+ homeostasis requirements of neurons.

Critical role of sodium in cytosolic [Ca2+] elevations in cultured hippocampal CA1 neurons during anoxic depolarization.

Although the extent of ischemic brain damage is directly proportional to the duration of anoxic depolarization (AD), the mechanism of cytosolic [Ca2+] ([Ca2+]c) elevation during AD is poorly understood. To address the mechanism in this study, [Ca2+]c was monitored in cultured rat hippocampal CA1 neurons loaded with a Ca‐sensitive dye, fura‐2FF, and exposed to an AD‐simulating medium containing (in mmol/L): K+ 65, Na+ 50, Ca2+ 0.13, glutamate 0.1, and pH reduced to 6.6. Application of this medium promptly elevated [Ca2+]c to about 30 µmol/L, but only if oxygen was removed, the respiratory chain was inhibited, or if the mitochondria were uncoupled. These high [Ca2+]c elevations depended on external Ca2+ and could not be prevented by inhibiting NMDA or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)/kainate receptors, or gadolinium‐sensitive channels. However, they could be prevented by removing external Na+ or simultaneously inhibiting NMDA and AMPA/kainate receptors; 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB‐R7943), an inhibitor of plasmalemmal Na+/Ca2+ exchanger, partly suppressed them. The data indicate that the [Ca2+]c elevations to 30 µmol/L during AD result from Na+ influx. Activation of either NMDA or AMPA/kainate channels provides adequate Na+ influx to induce these [Ca2+]c elevations, which are mediated by KB‐R7943‐sensitive and KB‐R7943‐resistant mechanisms.

NCX and NCKX Operation in Ischemic Neurons

Abstract:  Within the first 2 min of global brain ischemia, extracellular [K+] ([K+]o) increases above 60 mM and [Na+]o drops to about 50 mM, indicating a massive K+ efflux and Na+ influx, a phenomenon known as anoxic depolarization (AD). Similar ionic shifts take place during repetitive peri‐infarct depolarizations (PID) in the area penumbra in focal brain ischemia. The size of ischemic infarct is determined by the duration of AD and PID. However, the mechanism of cytosolic [Ca2+] ([Ca2+]c) elevation during AD or PID is poorly understood. Our data show that the exposure of cultured rat hippocampal CA1 neurons to AD‐like conditions promptly elevates [Ca2+]c to about 30 μM. These high [Ca2+]c elevations depend on external Ca2+ and can be prevented by removing Na+ or by simultaneously inhibiting NMDA and AMPA/kainate receptors. These data indicate that [Ca2+]c elevations during AD result from Na+ influx via either NMDA or AMPA/kainate channels. The mechanism of the Na‐dependent [Ca2+]c elevations may involve a reversal of plasmalemmal Na+/Ca2+ (NCX) and/or Na+/Ca2++ K+ (NCKX) exchangers. KB‐R7943, an NCX inhibitor, suppresses a fraction of the Na‐dependent Ca2+ influx during AD. Therefore, Ca2+ influx via NCX and a KB‐R7943‐resistant pathway (possibly NCKX) is involved. Inhibition of the Na‐dependent Ca2+ influx is likely to decrease ischemic brain damage. No drugs are known that are able to inhibit the KB‐R7943‐resistant component of Na‐dependent Ca2+ influx during AD. The present data encourage development of such agents as potential therapeutic means to limit ischemic brain damage after stroke or heart attack.

Proton-dependent zinc release from intracellular ligands

In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn2+ into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn2+. Although exposure to the protonophore FCCP precludes reloading of the mitochondria and acidic organelles with Zn2+, FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn2+. Therefore, Zn2+‐releasing stores were not mitochondria or acidic organelles but rather intracellular Zn2+ ligands. To test which ligands might be involved, the rate of acid‐induced Zn2+ release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn2+] was monitored in vitro using the ratiometric Zn2+‐sensitive fluorescent probe FuraZin‐1. Carnosine failed to chelate Zn2+ but did chelate Cu2+; the remaining ligands chelated Zn2+ and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc–cysteine complexes rapidly accelerated the rate of Zn2+ release. The zinc–cysteine complexes also released Zn2+ when a histidine‐modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc–cysteine complexes can contain micromolar amounts of Zn2+, these complexes may represent the stores responsible for an acid‐induced intracellular Zn2+ release.