Ketamine causes psychotic episodes and is often used as pharmacological model of psychotic-like behavior in animals. There is increasing evidence that molecular mechanism of its action is more complicated than just N-methyl-D-aspartic acid (NMDA) receptor antagonism and involves interaction with the components of calcium homeostatic machinery, in particular plasma membrane calcium pump (PMCA). Therefore, in this study we aimed to characterize brain region-specific effects of ketamine on PMCA activity, interaction with NMDA receptor through postsynaptic density protein 95 (PSD95) scaffolding proteins and glutamate release from nerve endings. In our study, ketamine induced behavioral changes in healthy male rats consistent with psychotic effects. In the same animals, we were able to demonstrate significant inhibition of plasma membrane calcium ATPase (PMCA) activity in cerebellum, hippocampus and striatum. The expression level and isoform composition of PMCAs were also affected in some of these brain compartments, with possible compensatory effects of PMCA1 substituting for decreased expression of PMCA3. Expression of the PDZ domain-containing scaffold protein PSD95 was induced and its association with PMCA4 was higher in most brain compartments upon ketamine treatment. Moreover, increased PSD95/NMDA receptor direct interaction was also reported, strongly suggesting the formation of multiprotein complexes potentially mediating the effect of ketamine on calcium signaling. We further support this molecular mechanism by showing brain region-specific changes in PSD95/PMCA4 spatial colocalization. We also show that ketamine significantly increases synaptic glutamate release in cortex and striatum (without affecting total tissue glutamate content), inducing the expression of vesicular glutamate transporters and decreasing the expression of membrane glutamate reuptake pump excitatory amino acid transporters 2 (EAAT2). Thus, ketamine-mediated PMCA inhibition, by decreasing total Ca2+ clearing potency, may locally raise cytosolic Ca2+ promoting excessive glutamate release. Regional alterations in glutamate secretion can be further driven by PSD95-mediated spatial recruitment of signaling complexes including glutamate receptors and calcium pumps, representing a novel mechanism of psychogenic action of ketamine.

Several lines of evidence suggest the contribution of age-related decline in plasma membrane calcium pump (PMCA) to the onset of neurodegenerative diseases. From four PMCA isoforms, PMCA2, and PMCA3 respond to a rapid removal of Ca2+ and are expressed predominantly in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in differentiated PC12 cells accelerated cell differentiation, but increased apoptosis in PMCA2-deficient line. We also demonstrated that altered expression of voltage-dependent calcium channels correlated with their higher contribution to Ca2+ influx, which varied between PMCA-reduced lines. Here, we propose a mechanism unique for differentiated PC12 cells by which PMCA2 and PMCA3 regulate pGAP43/GAP43 ratio and the interaction between GAP43 and calmodulin (CaM). Although down-regulation of PMCA2 or PMCA3 altered the content of GAP43/pGAP43, of paramount importance for the regulatory mechanism is a disruption of isoform-specific inhibitory PMCA/calcineurin interaction. In result, higher endogenous calcineurin (CaN) activity leads to hypophosphorylation of GAP43 in PMCA2- or PMCA3-deficient lines and intensification of GAP43/CaM complex formation, thus potentially limiting the availability of free CaM. In overall, our results indicate that both “fast” PMCA isoforms could actively regulate the local CaN function and CaN-downstream processes. In connection with our previous observations, we also suggest a negative feedback of cooperative action of CaM, GAP43, and CaN on P/Q and L-type channels activity. PMCAs- and CaN-dependent mechanism presented here, may signify a protective action against calcium overload in neuronal cells during aging, as well a potential way for decreasing neuronal cells vulnerability to neurodegenerative insults.

Plasma membrane Ca2+-ATPase (PMCA) by extruding Ca2+ outside the cell, actively participates in the regulation of intracellular Ca2+ concentration. Acting as Ca2+/H+ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca2+ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pHmito and pHcyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca2+ clearance and partially attenuated cellular acidification during KCl-stimulated Ca2+ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca2+ overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca2+-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.

The aging process is a physiological phenomenon associated with progressive changes in metabolism, genes expression, and cellular resistance to stress. In neurons, one of the hallmarks of senescence is a disturbance of calcium homeostasis that may have far-reaching detrimental consequences on neuronal physiology and function. Among several proteins involved in calcium handling, plasma membrane Ca2+-ATPase (PMCA) is the most sensitive calcium detector controlling calcium homeostasis. PMCA exists in four main isoforms and PMCA2 and PMCA3 are highly expressed in the brain. The overall effects of impaired calcium extrusion due to age-dependent decline of PMCA function seem to accumulate with age, increasing the susceptibility to neurotoxic insults. To analyze the PMCA role in neuronal cells, we have developed stable transfected differentiated PC12 lines with down-regulated PMCA2 or PMCA3 isoforms to mimic age-related changes. The resting Ca2+ increased in both PMCA-deficient lines affecting the expression of several Ca2+-associated proteins, i.e., sarco/endoplasmic Ca2+-ATPase (SERCA), calmodulin, calcineurin, GAP43, CCR5, IP3Rs, and certain types of voltage-gated Ca2+ channels (VGCCs). Functional studies also demonstrated profound changes in intracellular pH regulation and mitochondrial metabolism. Moreover, modification of PMCAs membrane composition triggered some adaptive processes to counterbalance calcium overload, but the reduction of PMCA2 appeared to be more detrimental to the cells than PMCA3.

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Schizophrenia is a common debilitating disease characterized by continuous or relapsing episodes of psychosis. Although the molecular mechanisms underlying this psychiatric illness remain incompletely understood, a growing body of clinical, pharmacological, and genetic evidence suggests that G protein-coupled receptors (GPCRs) play a critical role in disease development, progression, and treatment. This pivotal role is further highlighted by the fact that GPCRs are the most common targets for antipsychotic drugs. The GPCRs activation evokes slow synaptic transmission through several downstream pathways, many of them engaging intracellular Ca2+ mobilization. Dysfunctions of the neurotransmitter systems involving the action of GPCRs in the frontal and limbic-related regions are likely to underly the complex picture that includes the whole spectrum of positive and negative schizophrenia symptoms. Therefore, the progress in our understanding of GPCRs function in the control of brain cognitive functions is expected to open new avenues for selective drug development. In this paper, we review and synthesize the recent data regarding the contribution of neurotransmitter-GPCRs signaling to schizophrenia symptomology.

Chronic N-methyl-D-aspartate receptor (NMDAR) antagonist treatment can provide valuable neurochemical and neuroanatomical models of experimental psychosis. One such antagonist, ketamine, with its short half-time and well-documented psychotomimetic action, has cognitive effects resembling various aspects of schizophrenia-like symptoms. In order to obtain insights into possible relationships between Ca2+ homeostasis and schizophrenia-related symptoms, we investigate the effects of chronic ketamine administration on intracellular Ca2+ levels in various brain regions and on the expression level of key members of the neuronal Ca2+-handling system in rats. We show increased intracellular [Ca2+] in all of the examined brain regions following ketamine treatment but an altered cytosolic Ca2+ level correlated with hyperlocomotor activity was only established for the cortex and striatum. Our findings also suggest that an imbalance in the expression between the calcium “on” and “off” systems contributes to the deregulation of brain Ca2+ homeostasis in our ketamine-induced model of experimental psychosis. Identification of the genes whose expression is affected by ketamine treatment indicates their involvement as putative etiological factors in schizophrenia.

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