Bioenergetic Regulation of Neuronal Vacuolar-type H+- ATPase

Abstract

Neuronal vacuolar-type H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to acidify intracellular organelles such as lysosomes and synaptic vesicles, creating a proton gradient by which neurotransmitters can enter the vesicle through proton-coupled neurotransmitter transporters, a crucial step in neurotransmission (Moriyama, Maeda, & Futai, 1992). It is composed of two reversible domains, the integral V0 that allows proton translocation and catalytic peripheral V1 that is responsible for ATP hydrolysis. The V-ATPase regulates its activity through a process called reversible disassembly. When the V0 and V1 domains assemble, V-ATPase is activated and allows the influx of protons. When the domains disassemble, V-ATPase is inactivated, and proton transport does not occur (Beltran & Nelson, 1992). V-ATPase assembly was previously demonstrated to be regulated by glucose in yeast (Kane, 1995) and some mammalian cells (Toei, Saum, & Forgac, 2010), but whether and how glucose regulates neuronal V-ATPase is unclear. This study investigates the effect of bioenergetic substrates on neuronal V-ATPase assembly. Neuro2a (N2a) cells were differentiated for 96 hours, glucose-deprived overnight, and then treated with substrates such as glucose, beta-hydroxybutyrate, sodium pyruvate, creatine phosphate, and creatine monohydrate for 20 minutes prior to cell lysate preparation. To study V-ATPase assembly, assembled V-ATPase were captured through co-immunoprecipitation. Equal amounts of cell lysate were incubated with V1 antibody-coupled resin. Immunoblotting was then performed on the eluate to detect the V1 and V0 domains. The density of the bands was quantified and the ratio of V0 and V1 domains was used to determine V0/ V1 assembly. The results demonstrate changes in glucose availability (deprivation/stimulation) did not impact V-ATPase assembly in differentiated N2a cells. Furthermore, the results show other bioenergetic substrates (pyruvate, beta-hydroxybutyrate, and creatine molecules) did not induce changes in neuronal V-ATPase assembly. Our results did not support the well-documented role of glucose regulation of V-ATPase assembly demonstrated in previous studies conducted in yeast and mammalian cells. Several pitfalls of this study may have contributed to the negative results. The glucose concentration for stimulation and incubation time for glucose-deprivation may not have been optimal for differentiated N2a cells; further investigation will be needed to optimize these conditions. The glucose concentration used for stimulation after overnight glucose-deprivation did not exceed the physiological glucose concentration; higher concentrations of glucose should be tested. Previous studies had various incubation times for glucose-deprivation that could be experimentally optimized. Furthermore, the experiments could be replicated in other neuronal cells or in primary neurons to validate the results.