The NHE family of ion exchangers includes six isoforms (NHE1-NHE6) that function in an electroneutral exchange of intracellular H(+) for extracellular Na(+). This review focuses on the only ubiquitously expressed isoform, NHE1, which is localized at the plasma membrane where it plays a critical role in intracellular pH (pHi) and cell volume homeostasis. All NHE isoforms share a similar topology: an N-terminus of 12 transmembrane (TM) alpha-helices that collectively function in ion exchange, and a C-terminal cytoplasmic regulatory domain that modulates transport activity by the TM domain. Extracellular signals, mediated by diverse classes of cell-surface receptors, regulate NHE1 activity through distinct signaling networks that converge to directly modify the C-terminal regulatory domain. Modifications in the C-terminus, including phosphorylation and the binding of regulatory proteins, control transport activity by altering the affinity of the TM domain for intracellular H(+). Recently, it was determined that NHE1 also functions as a membrane anchor for the actin-based cytoskeleton, independently of its role in ion translocation. Through its effects on pHi homeostasis, cell volume, and the actin cortical network, NHE1 regulates a number of cell behaviors, including adhesion, shape determination, migration, and proliferation.

It is well established that activation of the Na-H exchanger NHE1 and increases in intracellular pH (pH i ) are early and universal responses to mitogens and have permissive effects in promoting cell proliferation. Despite this evidence, a specific role for NHE1 or pH i in cell cycle progression remains undetermined. We now show that NHE1 activity and pH i regulate the timing of G 2 /M entry and transition. Prior to G 2 /M entry there is a rapid and transient increase in NHE1 activity and pH i , but in fibroblasts expressing a mutant NHE1 that lacks ion translocation activity, this increase in pH i is attenuated, S phase is delayed, and G 2 /M transition is impaired. In the absence of ion translocation by NHE1, expression of cyclin B1 and the kinase activity of Cdc2 are decreased and Wee1 kinase expression increases. Increasing pH i in the absence of NHE1 activity, however, is sufficient to restore Cdc2 activity and cyclin B1 expression and to promote G 2 /M entry and transition. These data indicate that a transient increase in pH i induced by NHE1 promotes the timing of G 2 /M, and they suggest that increases in pH i at the completion of S phase may constitute a previously unrecognized checkpoint for progression to G 2 and mitosis.The ubiquitously expressed plasma membrane Na-H exchanger NHE1 1 regulates intracellular pH (pH i ) homeostasis and has a permissive effect in promoting cell proliferation. Activation of NHE1 and increased pH i are early and universal responses to mitogenic stimulation (1). Growth factor-dependent cell proliferation is attenuated in NHE1-deficient cells (2, 3) in cells treated with pharmacological inhibitors of NHE1 (4 -6) and in cells expressing a mutant NHE1 that is deficient in ion translocation (7). Additionally, retrovirus-induced transformation requires an NHE1-dependent increase in pH ii , and clamping pH i to prevent alkalinization inhibits proliferation and a transformed phenotype (8). Despite an established role for NHE1 in mitogeninduced cell proliferation, the mechanisms whereby NHE1 activity and increased pH i promote cell proliferation are not well understood. We therefore investigated cell cycle progression in fibroblasts expressing a mutant NHE1 that selectively lacks ion translocation activity and is unable to regulate pH i . EXPERIMENTAL PROCEDURES Cell Preparation-LtkϪ and LAP1 fibroblasts were obtained from J. Pouyssegur (University of Nice, France) (9). LAP1 cells stably expressing wild-type and NHE1-266I were obtained by co-transfection of pRSV-neo (1.0 g) with NHE1 plasmids (10 g of pCMV-NHE1) as described previously (7). Cells were maintained in DMEM supplemented with 10% FCS (growth medium).NHE1 Activity and pH i -NHE1 activity was determined as described previously (10) in cells loaded with the acetoxy-methyl ester derivative of the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF, Molecular Probes). NHE1 activity was determined by measuring the rate of pH i recovery (dpH i /dt) from an NH 4 Cl-induced acid load by evaluating the derivative...

Background: In mammalian cells changes in intracellular pH (pH i ), which are predominantly controlled by activity of plasma membrane ion exchangers, regulate a diverse range of normal and pathological cellular processes. How changes in pH i affect distinct cellular processes has primarily been determined by evaluating protein activities and we know little about how pH i regulates gene expression.

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The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([Cl]i) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [Cl]i. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [Cl]i. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [Cl]i results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [Cl]i, as well as elevating [Cl]i, on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [Cl]i is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [Cl]i. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [Cl]i may participate in modulating outflow facility across the TM.

Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca 2؉ -binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signals, whether its nuclear and cytoplasmic localization is regulated and has distinct functions remain unknown. We show that CHP1 is predominantly in the nucleus in quiescent fibrobasts, is translocated to cytoplasmic compartments with growth medium, and that translocation is inhibited by mutations in the nuclear export motifs. In a screen for proteins co-precipitating with CHP1 in quiescent cells we identified the upstream binding factor UBF, a DNA-binding protein and component of the RNA polymerase I complex regulating RNA synthesis. The CHP1-UBF interaction is restricted to the nucleus and inhibited by Ca 2؉ . Nuclear retention of CHP1 attenuates the abundance of UBF in the nucleolus and inhibits RNA synthesis when quiescent cells are transferred to growth medium. These data show UBF as a newly identified CHP1-binding protein and regulation of RNA synthesis as a newly identified function for nuclear-localized CHP1, which is distinct from CHP1 functions in the cytosol.A protein that localizes in multiple subcellular compartments often has spatially distinct functions. One example is the calcineurin B homologous protein CHP1, 4 an N-myristoylated EF-hand Ca 2ϩ -binding protein sharing ϳ40% identity with the regulatory B subunit of the protein phosphatase calcineurin and ϳ30% identity with calmodulin. CHP1, also known as p22, is evolutionarily conserved in human, rat, and Caenorhabditis elegans, is ubiquitously expressed in mammalian tissues, and localizes at the plasma membrane and in vesicular, cytosolic, and nuclear compartments. We previously identified CHP1 in a screen for proteins interacting with the C-terminal cytoplasmic domain of the plasma membrane Na-H exchanger NHE1 (1). Subsequent studies found that CHP1 is a cofactor essential for NHE1 activity (2-4) and also interacts with the Na-H exchanger isoform NHE3 (5). In vesicular compartments, CHP1 associates with membranes of the early secretory pathway (6) and is required for membrane traffic in a cell-free assay (7). In the cytoplasm, CHP1 complexes with microtubules (8) to facilitate microtubule-membrane interactions (9). Microtubule association can be mediated by direct binding of CHP1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (6), which associates with the microtubule cytoskeleton within the early secretory pathway, or to the kinesin-related motor KIF1〉␤2 (10), which mediates the transport of synaptic vesicles in neurons. In the cytosolic compartment CHP1 also directly binds the calcineurin catalytic A subunit and inhibits calcineurin phosphatase activ...