Activation of AMP-activated protein kinase regulates hippocampal neuronal pH by recruiting Na(+)/H(+) exchanger NHE5 to the cell surface.

Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H(+)-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na(+)/H(+) exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.

CK2 phosphorylation of an acidic Ser/Thr di-isoleucine motif in the Na+/H+ exchanger NHE5 isoform promotes association with beta-arrestin2 and endocytosis.

Internalization of the Na(+)/H(+) exchanger NHE5 into recycling endosomes is enhanced by the endocytic adaptor proteins ß-arrestin1 and -2, best known for their preferential recognition of ligand-activated G protein-coupled receptors (GPCRs). However, the mechanism underlying their atypical association with non-GPCRs, such as NHE5, is unknown. In this study, we identified a highly acidic, serine/threonine-rich, di-isoleucine motif (amino acids 697-723) in the cytoplasmic C terminus of NHE5 that is recognized by ß-arrestin2. Gross deletions of this site decreased the state of phosphorylation of NHE5 as well as its binding and responsiveness to ß-arrestin2 in intact cells. More refined in vitro analyses showed that this site was robustly phosphorylated by the acidotropic protein kinase CK2, whereas other kinases, such as CK1 or the GPCR kinase GRK2, were considerably less potent. Simultaneous mutation of five Ser/Thr residues within 702-714 to Ala ((702)ST/AA(714)) abolished phosphorylation and binding of ß-arrestin2. In transfected cells, the CK2 catalytic α subunit formed a complex with NHE5 and decreased wild-type but not (702)ST/AA(714) NHE5 activity, further supporting a regulatory role for this kinase. The rate of internalization of (702)ST/AA(714) was also diminished and relatively insensitive to overexpression of ß-arrestin2. However, unlike in vitro, this mutant retained its ability to form a complex with ß-arrestin2 despite its lack of responsiveness. Additional mutations of two di-isoleucine-based motifs (I697A/L698A and I722A/I723A) that immediately flank the acidic cluster, either separately or together, were required to disrupt their association. These data demonstrate that discrete elements of an elaborate sorting signal in NHE5 contribute to ß-arrestin2 binding and trafficking along the recycling endosomal pathway.

Impact of anesthetic agents on cerebrovascular physiology in children.

The role of the pediatric neuroanesthetist is to provide comprehensive care to children with neurologic pathologies. The cerebral physiology is influenced by the developmental stage of the child. The understanding of the effects of anesthetic agents on the physiology of cerebral vasculature in the pediatric population has significantly increased in the past decade allowing a more rationale decision making in anesthesia management. Although no single anesthetic technique can be recommended, sound knowledge of the principles of cerebral physiology and anesthetic neuropharmacology will facilitate the care of pediatric neurosurgical patients.

beta-Arrestins bind and decrease cell-surface abundance of the Na+/H+ exchanger NHE5 isoform.

The neuronal Na(+)/H(+) exchanger NHE5 isoform not only resides in the plasma membrane but also accumulates in recycling vesicles by means of clathrin-mediated endocytosis. To further investigate the underlying molecular mechanisms, a human brain cDNA library was screened for proteins that interact with the cytoplasmic C-terminal region of NHE5 by using yeast two-hybrid methodology. One candidate cDNA identified by this procedure encoded beta-arrestin2, a specialized adaptor/scaffolding protein required for internalization and signaling of members of the G protein-coupled receptor superfamily. Direct interaction between the two proteins was demonstrated in vitro by GST fusion protein pull-down assays. Sequences within the N-terminal receptor activation-recognition domain and the C-terminal secondary receptor-binding domain of beta-arrestin2 conferred strong binding to the C terminus of NHE5. Full-length NHE5 and beta-arrestin2 also associated in intact cells, as revealed by their coimmunoprecipitation from extracts of transfected CHO cells. Moreover, ectopic expression of both proteins caused a redistribution of beta-arrestin2 from the cytoplasm to vesicles containing NHE5, and significantly decreased the abundance of the transporter at the cell surface. Comparable results were also obtained for the beta-arrestin1 isoform. These data reveal a broader role for arrestins in the trafficking of integral plasma membrane proteins than previously recognized.

Clathrin-mediated endocytosis and recycling of the neuron-specific Na+/H+ exchanger NHE5 isoform. Regulation by phosphatidylinositol 3'-kinase and the actin cytoskeleton.

Mammalian Na+/H+ exchangers (NHEs) are a family of integral membrane proteins that play central roles in sodium, acid-base, and cell volume homeostasis. The recently cloned NHE5 isoform is expressed predominantly in brain, but its functional and cellular properties are poorly understood. To facilitate its characterization, an epitope-tagged construct of NHE5 was ectopically expressed in nonneuronal and neuronal cells. In NHE-deficient Chinese hamster ovary AP-1 cells, NHE5 localized at the plasmalemma, but a significant fraction accumulated intracellularly in vesicles that concentrated in a juxtanuclear region. Similarly, in nerve growth factor-differentiated neuroendocrine PC12 cells and primary hippocampal neurons, immunolabeling of NHE5 was detected in endomembrane vesicles in the perinuclear region of the cell body but also along the processes. More detailed characterization in AP-1 cells using organelle-specific markers showed that NHE5 co-localized with internalized transferrin, a marker of recycling endosomes. Transient transfection of a dominant negative mutant of dynamin-1, which inhibits clathrin-mediated endocytosis, blocked uptake of transferrin as well as internalization of NHE5. Likewise, wortmannin inhibition of phosphatidylinositol 3'-kinase, a lipid kinase implicated in endosomal traffic, induced coalescence of vesicles containing NHE5 and caused a pronounced inhibition of plasmalemmal Na+/H+ exchange. By contrast, disruption of the F-actin cytoskeleton with cytochalasin D increased cell surface NHE5 activity and abundance. These observations demonstrate that NHE5 is localized to the recycling endosomal pathway and is dynamically regulated by phosphatidylinositol 3'-kinase and by the state of F-actin assembly.