Analysis of Ion Interactions with the K+ -dependent Na+/Ca+ Exchangers NCKX2, NCKX3, and NCKX4 IDENTIFICATION OF THR-551 AS A KEY RESIDUE IN DEFINING THE APPARENT K+ AFFINITY OF NCKX2

K(+)-dependent Na(+)/Ca(2+) exchangers (NCKX) catalyze cytosolic Ca(2+) extrusion and are particularly important for neuronal Ca(2+) signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca(2+) and Na(+) were similar, NCKX3 and -4 displayed approximately 40-fold higher affinities for K(+) ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K(+) affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca(2+) affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K(+) and Ca(2+) affinities were reduced. These results indicate that the apparently low K(+) affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K(+) liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K(+) binding pocket, because it was not possible to recreate the K(+)-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca(2+) signaling

Synthesis and Pharmacological Evaluation of a Novel Peptide Based on Anemonia sulcata BDS-I Toxin as a New KV3.4 Inhibitor Exerting a Neuroprotective Effect Against Amyloid-β Peptide

There is increasing evidence that the fast-inactivating potassium current IA, encoded by KV3. 4 channels, plays an important role in Alzheimer's Disease (AD), since the neurotoxic β-amyloid peptide1-42 (Aβ1-42) increases the IA current triggering apoptotic processes. The specific inhibition of KV3.4 by the marine toxin extracted from Anemonia sulcata, named blood depressing substance-I (BDS-I), reverts the Aβ peptide-induced cell death. The aim of the present study was to identify the smallest fragments of BDS-I, obtained by peptide synthesis, able to inhibit KV3.4 currents. For this purpose, whole-cell patch clamp technique was used to evaluate the effects of BDS-I fragments on KV3.4 currents in CHO cells heterologously expressing KV3.4. We found that BDS-I[1-8] fragment, containing the N-terminal octapeptide sequence of full length BDS-I, was able to inhibit KV3.4 currents in a concentration dependent manner, whereas the scrambled sequence of BDS-I[1-8] and all the other fragments obtained from BDS-I full length were ineffective. As we demonstrated in a previous study, BDS-I full length is able to counteract Aβ1-42-induced enhancement of KV3.4 activity, preventing Aβ1-42-induced caspase-3 activation and the abnormal nuclear morphology in NGF-differentiated PC-12 cells. Similarly to BDS-I, we found that BDS-I[1-8] blocking KV3.4 currents prevented Aβ1-42-induced caspase-3 activation and apoptotic processes. Collectively, these results suggest that BDS-I[1-8] could represent a lead compound to be developed as a new drug targeting KV3.4 channels

K+-Dependent Na+/Ca2+ Exchanger Isoform 2, Nckx2, Takes Part in the Neuroprotection Elicited by Ischemic Preconditioning in Brain Ischemia

Sodium/Calcium exchangers are neuronal plasma membrane antiporters which, by coupling Ca2+ and Na+ fluxes across neuronal membranes, play a relevant role in brain ischemia. The most brain-expressed isoform among the members of the K+-dependent Na+/Ca2+ exchanger family, NCKX2, is involved in the progression of the ischemic lesion, since both its knocking-down and its knocking-out worsens ischemic damage. The aim of this study was to elucidate whether NCKX2 functions as an effector in the neuroprotection evoked by ischemic preconditioning. For this purpose, we investigated: (1) brain NCKX2 expression after preconditioning and preconditioning + ischemia; (2) the contribution of AKT and calpain to modulating NCKX2 expression during preconditioning; and (3) the effect of NCKX2 knocking-out on the neuroprotection mediated by ischemic preconditioning. Our results showed that NCKX2 expression increased in those brain regions protected by ischemic preconditioning. These changes were p-AKT-mediated since its inhibition prevented NCKX2 up-regulation. More interestingly, NCKX2 knocking-out significantly prevented the protection exerted by ischemic preconditioning. Overall, our results suggest that NCKX2 plays a fundamental role in the neuroprotective effect mediated by ischemic preconditioning and support the idea that the enhancement of its expression and activity might represent a reasonable strategy to reduce infarct extension after stroke

nuclear encoded ncx3 and akap121 two novel modulators of mitochondrial calcium efflux in normoxic and hypoxic neurons

Abstract Mitochondria are highly dynamic organelles extremely important for cell survival. Their structure resembles that of prokaryotic cells since they are composed with two membranes, the inner (IMM) and the outer mitochondrial membrane (OMM) delimitating the intermembrane space (IMS) and the matrix which contains mitochondrial DNA (mtDNA). This structure is strictly related to mitochondrial function since they produce the most of the cellular ATP through the oxidative phosphorylation which generate the electrochemical gradient at the two sides of the inner mitochondrial membrane an essential requirement for mitochondrial function. Cells of highly metabolic demand like those composing muscle, liver and brain, are particularly dependent on mitochondria for their activities. Mitochondria undergo to continual changes in morphology since, they fuse and divide, branch and fragment, swell and extend. Importantly, they move throughout the cell to deliver ATP and other metabolites where they are mostly required. Along with the capability to control energy metabolism, mitochondria play a critical role in the regulation of many physiological processes such as programmed cell death, autophagy, redox signalling, and stem cells reprogramming. All these phenomena are regulated by Ca2+ ions within this organelle. This review will discuss the molecular mechanisms regulating mitochondrial calcium cycling in physiological and pathological conditions with particular regard to their impact on mitochondrial dynamics and function during ischemia. Particular emphasis will be devoted to the role played by NCX3 and AKAP121 as new molecular targets for mitochondrial function and dysfunction

Models and methods for conditioning the ischemic brain

Abstract Background In the last decades the need to find new neuroprotective targets has addressed the researchers to investigate the endogenous molecular mechanisms that brain activates when exposed to a conditioning stimulus. Indeed, conditioning is an adaptive biological process activated by those interventions able to confer resistance to a deleterious brain event through the exposure to a sub-threshold insult. Specifically, preconditioning and postconditioning are realized when the conditioning stimulus is applied before or after, respectively, the harmul ischemia. Aims and Results The present review will describe the most common methods to induce brain conditioning, with particular regards to surgical, physical exercise, temperature-induced and pharmacological approaches. It has been well recognized that when the subliminal stimulus is delivered after the ischemic insult, the achieved neuroprotection is comparable to that observed in models of ischemic preconditioning. In addition, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone. Conclusions The last decades have provided fascinating insights into the mechanisms and potential application of strategies to induce brain conditioning. Since the identification of intrinsic cell‐survival pathways should provide more direct opportunities for translational neuroprotection trials, an accurate examination of the different models of preconditioning and postconditioning is mandatory before starting any new project

Rebound effects of NCX3 pharmacological inhibition: A novel strategy to accelerate myelin formation in oligodendrocytes.

Abstract The Na+/Ca2+ exchanger NCX3 is an important regulator of sodium and calcium homeostasis in oligodendrocyte lineage. To date, no information is available on the effects resulting from prolonged exposure to NCX3 blockers and subsequent drug washout in oligodendroglia. Here, we investigated, by means of biochemical, morphological and functional analyses, the pharmacological effects of the NCX3 inhibitor, the 5–amino‐N‐butyl‐2–(4–ethoxyphenoxy)-benzamide hydrochloride (BED), on NCXs expression and activity, as well as intracellular [Na+]i and [Ca2+]i levels, during treatment and following drug washout both in human MO3.13 oligodendrocytes and rat primary oligodendrocyte precursor cells (OPCs). BED exposure antagonized NCX activity, induced OPCs proliferation and [Na+]i accumulation. By contrast, 2 days of BED washout after 4 days of treatment significantly upregulated low molecular weight NCX3 proteins, reversed NCX activity, and increased intracellular [Ca2+]i. This BED-free effect was accompanied by an upregulation of NCX3 expression in oligodendrocyte processes and accelerated expression of myelin markers in rat primary oligodendrocytes. Collectively, our findings show that the pharmacological inhibition of the NCX3 exchanger with BED blocker maybe followed by a rebound increase in NCX3 expression and reversal activity that accelerate myelin sheet formation in oligodendrocytes. In addition, they indicate that a particular attention should be paid to the use of NCX inhibitors for possible rebound effects, and suggest that further studies will be necessary to investigate whether selective pharmacological modulation of NCX3 exchanger may be exploited to benefit demyelination and remyelination in demyelinating diseases

Amyloid β-Induced Upregulation of Nav1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer's Disease Mouse Model

Hyperexcitability and alterations in neuronal networks contribute to cognitive impairment in Alzheimer's Disease (AD). Voltage-gated sodium channels (NaV), which are crucial for regulating neuronal excitability, have been implicated in AD-related hippocampal hyperactivity and higher incidence of spontaneous non-convulsive seizures. Here, we show by using primary hippocampal neurons exposed to amyloid-β1-42 (Aβ1-42) oligomers and from Tg2576 mouse embryos, that the selective upregulation of NaV1.6 subtype contributes to membrane depolarization and to the increase of spike frequency, thereby resulting in neuronal hyperexcitability. Interestingly, we also found that NaV1.6 overexpression is responsible for the aberrant neuronal activity observed in hippocampal slices from 3-month-old Tg2576 mice. These findings identify the NaV1.6 channels as a determinant of the hippocampal neuronal hyperexcitability induced by Aβ1-42 oligomers. The selective blockade of NaV1.6 overexpression and/or hyperactivity might therefore offer a new potential therapeutic approach to counteract early hippocampal hyperexcitability and subsequent cognitive deficits in the early stages of AD

Neuroprotective coordination of cell mitophagy by the ATPase Inhibitory Factor 1

The mitochondrial ATPase Inhibitory Factor 1 (hereafter referred to as IF1) blocks the reversal of the F1Fo-ATPsynthase to prevent detrimental consumption of cellular ATP and associated demise. Herein, we infer further its molecular physiology by assessing its protective function in neurons during conditions of challenged homeostatic respiration. By adopting in vitro and in vivo protocols of hypoxia/ischemia and re-oxygenation, we show that a shift in the IF1:F1Fo-ATPsynthase expression ratio occurs in neurons. This increased IF1 level is essential to induce accumulation of the PTEN-induced putative kinase 1 (PINK-1) and recruitment of the mitophagic ubiquitin ligase PARK-2 to promote autophagic “control” of the mitochondrial population. In IF1 overexpressing neurons ATP depletion is reduced during hypoxia/ischemia and the mitochondrial membrane potential (ΔYm) resilient to re-oxygenation as well as resistant to electrogenic, Ca2+ dependent depolarization. These data suggest that in mammalian neurons mitochondria adapt to respiratory stress by upregulating IF1, which exerts a protective role by coordinating pro-survival cell mitophagy and bioenergetics resilience

Decreased subunit stability as a novel mechanism for potassium current impairment by a KCNQ2 C terminus mutation causing benign familial neonatal convulsions.

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates

A Novel Hyperekplexia-causing Mutation in the Pre-transmembrane Segment 1 of the Human Glycine Receptor α1 Subunit Reduces Membrane Expression and Impairs Gating by Agonists

In this study, we have compared the functional consequences of three mutations (R218Q, V260M, and Q266H) in the alpha(1) subunit of the glycine receptor (GlyRA1) causing hyperekplexia, an inherited neurological channelopathy. In HEK-293 cells, the agonist EC(50s) for glycine-activated Cl(-) currents were increased from 26 microm in wtGlyRA1, to 5747, 135, and 129 microm in R218Q, V260M, and Q266H GlyRA1 channels, respectively. Cl(-) currents elicited by beta-alanine and taurine, which behave as agonists at wtGlyRA1, were decreased in V260M and Q266H mutant receptors and virtually abolished in GlyRA1 R218Q receptors. Gly-gated Cl(-) currents were similarly antagonized by low concentrations of strychnine in both wild-type (wt) and R218Q GlyRA1 channels, suggesting that the Arg-218 residue plays a crucial role in GlyRA1 channel gating, with only minor effects on the agonist/antagonist binding site, a hypothesis supported by our molecular model of the GlyRA1 subunit. The R218Q mutation, but not the V260M or the Q266H mutation, caused a marked decrease of receptor subunit expression both in total cell lysates and in isolated plasma membrane proteins. This decreased expression does not seem to explain the reduced agonist sensitivity of GlyRA1 R218Q channels since no difference in the apparent sensitivity to glycine or taurine was observed when wtGlyRA1 receptors were expressed at levels comparable with those of R218Q mutant receptors. In conclusion, multiple mechanisms may explain the dramatic decrease in GlyR function caused by the R218Q mutation, possibly providing the molecular basis for its association with a more severe clinical phenotype