The carrier-mediated, electroneutral exchange of Na+ for H+ across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na+/H+ exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP2) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP2, in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP2-binding motifs. Fusion proteins encoding these motifs bound PIP2 in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP2-binding domains had greatly reduced transport capability, implying that association with PIP2 is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP2.

To isolate a cDNA encoding Na ؉ /H ؉ exchanger isoform 5 (NHE5), we screened a human spleen library using exon sequences of the NHE5 gene. Clones spanning 2.9 kilobase pairs were isolated; however, they contained several introns and were missing coding sequences at both the 5 and 3 ends. The missing 5 sequences were obtained by 5-rapid amplification of cDNA ends and by analysis of an NHE5 genomic clone, and the missing 3 sequences were obtained by 3-rapid amplification of cDNA ends. Polymerase chain reaction amplification of brain cDNA yielded products in which each of the introns had been correctly excised, whereas the introns were retained in products from spleen and testis, suggesting that the NHE5 transcripts expressed in these organs do not encode a functional transporter. The intron/exon organization of the NHE5 gene was analyzed and found to be very similar to that of the NHE3 gene. The NHE5 cDNA, which encodes an 896-amino acid protein that is most closely related to NHE3, was expressed in Na ؉ /H ؉ exchanger-deficient fibroblasts and shown to mediate Na ؉ /H ؉ exchange activity. Northern blot analysis demonstrated that the mRNA encoding NHE5 is expressed in multiple regions of the brain, including hippocampus, consistent with the possibility that it regulates intracellular pH in hippocampal and other neurons.

Calcineurin B homologous protein (CHP) 1 and 2 are Ca 2؉ -binding proteins that modulate several cellular processes, including cytoplasmic pH by positively regulating plasma membrane-type Na ؉ /H ؉ exchangers (NHEs). Recently another CHP-related protein, termed tescalcin or CHP3, was also shown to interact with the ubiquitous NHE1 isoform, but seemingly suppressed its activity. However, the precise physical and functional nature of this association was not examined in detail. In this study, biochemical and cellular studies were undertaken to further delineate this relationship. Glutathione S-transferase-NHE1 fusion protein pulldown assays revealed that full-length CHP3 binds directly to the proximal juxtamembrane C-terminal region (amino acids 505-571) of rat NHE1 in the same region that binds CHP1 and CHP2. The interaction was further validated by coimmunoprecipitation and coimmunolocalization experiments using full-length CHP3 and wild-type NHE1 in transfected Chinese hamster ovary AP-1 cells. Simultaneous mutation of four hydrophobic residues within this region ( 530 FLDHLL 535 ) to either Ala, Gln, or Arg (FL-A, FL-Q, or FL-R) abrogated this interaction both in vitro and in intact cells. The NHE1 mutants were sorted properly to the cell surface but showed markedly reduced (FL-A) or minimal (FL-R and FL-Q) activity. Interestingly, and contrary to an earlier finding, ectopic coexpression of CHP3 up-regulated the cell surface activity of wild-type NHE1. This stimulation was not observed with the CHP3 binding-defective mutants. Mechanistically, overexpression of CHP3 did not alter the H ؉ sensitivity of wild-type NHE1 but rather promoted its biosynthetic maturation and half-life at the cell surface, thereby increasing the steady-state abundance of functional NHE1 protein. Monovalent cations such as Liϩ , Na ϩ , and K ϩ are transported across biological membranes in exchange for H ϩ by a family of alkali cation/proton countertransporters, commonly referred to as Na ϩ /H ϩ exchangers (NHE) 2 or antiporters. Phylogenetic analyses and functional studies have revealed the existence of at least 11 mammalian NHE isoforms that display varied primary structure (ϳ13-70% identity), tissue distribution, subcellular compartmentalization, cation selectivity, and function (1-3). Structurally, the NHEs are composed of two major domains as follows: an N terminus that contains 12 predicted membrane-spanning segments responsible for cation permeation and a C terminus that faces the cytoplasm and serves to regulate transport activity, membrane targeting, anchorage to the underlying actin cytoskeleton, and as a scaffold for the assembly of other signaling complexes (1, 2, 4 -7). Of these isoforms, NHE1 has received considerable attention because it is widely expressed and plays a vital role in several physiological processes, notably cytoplasmic pH homeostasis and maintenance of cell volume, but also cell shape, migration, proliferation, differentiation, and apoptosis (7-13). Accordingly, diverse signals (e.g. hormones, mitogens, an...

Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (≥10 dynes/cm2). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.

Axon degeneration is a critical pathological feature of many neurodegenerative diseases. Misregulation of local axonal ion homeostasis has been recognized as an important yet understudied mechanism for axon degeneration. Here we report a chemically induced, recessive mouse mutation, vacillator (vac), which causes ataxia and concomitant axon degeneration of cerebellar Purkinje cells. By positional cloning, we identified vac as a point mutation in the calcineurin-like EF hand protein 1 (Chp1) gene that resulted in the production of mutant CHP1 isoforms with an amino acid substitution in a functional EF-hand domain or a truncation of this motif by aberrant splicing and significantly reduced protein levels. CHP1 has been previously shown to interact with the sodium hydrogen exchanger 1 (NHE1), a major regulator of intracellular pH. We demonstrated that CHP1 assists in the full glycosylation of NHE1 that is necessary for the membrane localization of this transporter and that truncated isoforms of CHP1 were defective in stimulating NHE1 biosynthetic maturation. Consistent with this, membrane localization of NHE1 at axon terminals was greatly reduced in Chp1-deficient Purkinje cells before axon degeneration. Furthermore, genetic ablation of Nhe1 also resulted in Purkinje cell axon degeneration, pinpointing the functional convergence of the two proteins. Our findings clearly demonstrate that the polarized presynaptic localization of NHE/CHP1 is an important feature of neuronal axons and that selective disruption of NHE1-mediated proton homeostasis in axons can lead to degeneration, suggesting that local regulation of pH is pivotal for axon survival.