Glutamate-Induced ATP Synthesis: Relationship between Plasma Membrane Na⁺/Ca²⁺Exchanger and Excitatory Amino Acid Transporters in Brain and Heart Cell Models

Abstract

It is known that glutamate (Glu), the major excitatory amino acid in the central nervous system, can be an essential source for cell energy metabolism. Here we investigated the role of the plasma membrane Na 1 /Ca 21 exchanger (NCX) and the excitatory amino acid transporters (EAATs) in Glu uptake and recycling mechanisms leading to ATP synthesis. We used different cell lines, such as SH-SY5Y neuroblastoma, C6 glioma and H9c2 as neuronal, glial, and cardiac models, respectively. We first observed that Glu increased ATP production in SH-SY5Y and C6 cells. Pharmacological inhibition of either EAAT or NCX counteracted the Glu-induced ATP synthesis. Furthermore, Glu induced a plasma membrane depolarization and an intracellular Ca 21 increase, and both responses were again abolished by EAAT and NCX blockers. In line with the hypothesis of a mutual interplay between the activities of EAAT and NCX, coimmunoprecipitation studies showed a physical interaction between them. We expanded our studies on EAAT/NCX interplay in the H9c2 cells. H9c2 expresses EAATs but lacks endogenous NCX1 expression. Glu failed to elicit any significant response in terms of ATP synthesis, cell depolarization, and Ca 21 increase unless a functional NCX1 was introduced in H9c2 cells by stable transfection. Moreover, these responses were counteracted by EAAT and NCX blockers, as observed in SH-SY5Y and C6 cells. Collectively, these data suggest that plasma membrane EAAT and NCX are both involved in Glu-induced ATP synthesis, with NCX playing a pivotal role.

Astroglial glutamate transporters coordinate excitatory signaling and brain energetics

“…A variety of hypothesis-driven or yeast two-hybrid approaches were used to identify proteins that co-immunoprecipitate with one or more of the Na + -dependent glutamate transporters, including Ajuba (Marie et al, 2002), glutamate transporter associated proteins (GTRAPs) (Jackson et al, 2001; Lin et al, 2001), protein kinase Cα (González et al, 2003; González et al, 2005), a septin GTPase (Sept 2) (Kinoshita et al, 2004), syntaxin 1A (Yu et al, 2006), PSD-95 (Gonzalez-Gonzalez et al, 2008; Gonzalez-Gonzalez et al, 2009), the actin binding protein α-adducin (Bianchi et al, 2010), Na + /H + exchanger regulatory proteins (Lee et al, 2007; Sato et al, 2013), protein interacting with C kinase (PICK1) (Bassan et al, 2008), membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) (Zou et al, 2011), a glutamine transporter (SNAT3) (Martinez-Lozada et al, 2013), and the sodium/calcium exchanger (NCX1) (Magi et al, 2012; Magi et al, 2013). Many of these proteins directly or indirectly regulate various aspects of transporter function, including maturation, targeting, trafficking, or functional coupling of these transporters.…”

“…The reversal potential of the NCX in astrocytes is close to the resting membrane potential (Kirischuk et al, 1997; Reyes et al, 2012), such that NCX can rapidly operate in the so-called reverse mode (Na + out/Ca 2+ in) in response to small Na + loads such as occur with glutamate transport (Kirischuk et al, 1997; Langer and Rose, 2009; Reyes et al, 2012; Rojas et al, 2013). In astrocyte cultures or in organotypic hippocampal slice cultures, NCX appears to operate predominately in the reversed mode even under resting conditions (Jackson and Robinson, 2015; Magi et al, 2013; Reyes et al, 2012; Rojas et al, 2013) and thus appears to control basal [Ca 2+ ] in astrocytes. This control of [Ca 2+ ] i may be direct (via NCX) or indirect via Ca 2+ -induced Ca 2+ -release from intracellular Ca 2+ stores (Lencesova et al, 2004).…”

Neuronal Activity and Glutamate Uptake Decrease Mitochondrial Mobility in Astrocytes and Position Mitochondria Near Glutamate Transporters

“…In astrocytes, activation of glutamate transport activates the reverse mode of the NCX subsequent to Na ϩ entry, resulting in an increase in intracellular [Ca 2ϩ ] (Magi et al, 2013;Rojas et al, 2013). We tested the hypothesis that reversal of the NCX contributes to immobilization of mitochondria in astrocytes.…”

GLAST But Not Least—Distribution, Function, Genetics and Epigenetics of l-Glutamate Transport in Brain—Focus on GLAST/EAAT1

“…GLAST was found to be present and probably functional at the mammalian neuromuscular junction [45], in the choroid plexus [46] and in other tissues, for example in pancreas [47] and in the heart muscle cells where it may have an important function [48]. GLT and possibly other EAATs exist in testes [49,50] and EAAT5 is present in many peripheral tissues [42,51].…”