Cellular localization and functional characterization of brain enriched sodium proton exchanger NHE5

The central nervous system is extremely sensitive to global and local changes in pH, which are important to neural survival and excitability. Ion transporters that move acid-base equivalents regulate cellular pH and can therefore influence neuronal homeostasis. The SLC9a family of mammalian alkali cation-proton exchangers, commonly known as sodium-proton exchangers (NHE), are transmembrane proteins that normally catalyze the electroneutral exchange of extracellular sodium (and/or potassium) for intracellular protons. Thus, NHE contribute to the regulation of intracellular pH and volume homeostasis. The NHE family consists of eleven isoforms that have differential tissue distributions, subcellular locations and regulatory functions. Within the nervous system, many of the NHE isoforms can be detected. However, mRNA of the fifth isoform of the exchanger family demonstrates an enriched expression in nervous tissues unlike any other NHE isoform.;Despite the unique localization of NHE5, defining a precise physiological role for the exchanger has proven difficult. More information about the regulation of NHE5 would provide insight into the role of the exchanger in nervous tissue. Moreover, its protein expression amongst brain tissues has not been confirmed, nor has the subcellular distribution of the exchanger, although this information could suggest its endogenous functions. To elucidate the localization of NHE5, a highly selective and specific rabbit polyclonal antibody was generated. This antibody was used to determine the subcellular localization of NHE5, which partially colocalized with the transferrin receptor in primary neuronal cultures. Electron microscopy of immune-labelled cortical and hippocampal sections revealed that NHE5 can be found close to synapses and in the soma of different neural cells. Using an immunocytochemical and pharmacological approach, the presence and activity of NHE5 was detected in glial cells.;In addition to the lack of information pertaining to NHE5 localization, little is known about the function and regulation of this exchanger compared to its homologues NHE1 and NHE3. Considering the high metabolic rates of brain tissues and the acidic nature of the by-products of metabolism, it was hypothesized that NHE5 may be necessary to regulate cytosolic pH. This hypothesis was supported by the finding that the metabolic sensor, AMPK, interacts with NHE5, as determined by a yeast two-hybrid screen. The activation of AMPK by metabolic stressors stimulated heterologously expressed NHE5 activity at the plasma membrane through phosphorylation of specific serine sites in the C-terminal tail of the exchanger. The observable increase in NHE5 activity was the result of its redistribution to the cell surface rather than a change in its kinetic properties. The observations in cell culture were mimicked in primary hippocampal cultures, where pharamacological activation of AMPK and metabolic inhibition induced a greater abundance of NHE5 in close proximity to the cell surface.;Finally, the results of the aforementioned yeast two-hybrid screen yielded another interacting partner, GAPDH, which plays a key role during glycolysis. Inhibition of GAPDH enzymatic activity and cellular metabolic stress both increased the association between GAPDH and NHE5, and also increased the surface abundance of NHE5, and its catalytic activity.;These data demonstrate the subcellular and cell-type specific localization of endogenous NHE5 protein and provide evidence for a novel physiological role of NHE5 in maintaining cell metabolic status and reveals AMPK and GAPDH as novel regulators of the brain-enriched exchanger.