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The Ca2+-dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including theSaccharomyces cerevisiaevacuolar Ca2+/H+ exchanger Vcx1p. In yeast and plants, cation/H+ exchangers are important in shaping cytosolic Ca2+ levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca2+, Mn2+ and Cd2+. Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca2+ transporter and may also function in Cd2+ transport, but cannot transport Mn2+. Furthermore, direct Ca2+ transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele ofVCX1(VCX1-S204A/L208P), termedVCX1-M1, was previously found to confer strong Mn2+ tolerance. Here we demonstrate that this Mn2+ tolerance is independent of the Ca2+/Mn2+-ATPase Pmr1p and results from Mn2+-specific vacuolar transport activity of Vcx1-M1p. This Mn2+ transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn2+ tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p. [ABSTRACT FROM AUTHOR]

Examines manganese specificity determinants in the Arabidopsis metal/H[sup +] antiporter CAX2. Construction of CAX1/CAX2 chimeras; Analysis of the CAX2 c-domain; Provision of evidence for the feasibility of altering substrate specificity in a metal/H[sup +] antiporter, a family of transporters found in a variety of organisms.

Describes the preliminary localization of Cation Exchanger1 (cax1) to the tonoplast and the molecular characterization of cax1 mutants of Arabidopsis. Properties exhibited by cax1 mutants; Perturbation of ion homeostasis in cax1 mutants; Morphological phenotypes of cax1 alleles.

Ca2+ levels in plants are controlled in part by H+/Ca2+ exchangers. Structure/function analysis of the Arabidopsis H+/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca2+ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca2+ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca2+ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca2+ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca2+ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca2+ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca2+ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca2+ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues. [ABSTRACT FROM AUTHOR]

Betulinic acid (BA) is a major constituent of Zizyphus seeds that have been long used as therapeutic agents for sleep-related issues in Asia. BA is a pentacyclic triterpenoid. It also possesses various anti-cancer and anti-inflammatory effects. Current commercially available sleep aids typically use GABAergic regulation, for which many studies are being actively conducted. However, few studies have focused on acetylcholine receptors that regulate wakefulness. In this study, we utilized BA as an antagonist of α3β4 nicotinic acetylcholine receptors (α3β4 nAChRs) known to regulate rapid-eye-movement (REM) sleep and wakefulness. Effects of BA on α3β4 nAChRs were concentration-dependent, reversible, voltage-independent, and non-competitive. Site-directed mutagenesis and molecular-docking studies confirmed the binding of BA at the molecular level and showed that the α3 subunit L257 and the β4 subunit I263 residues affected BA binding. These data demonstrate that BA can bind to a binding site different from the site for the receptor's ligand, acetylcholine (ACh). This suggests that BA may be an effective antagonist that is unaffected by large amounts of ACh released during wakefulness and REM sleep. Based on the above experimental results, BA is likely to be a therapeutically useful sleep aid and sedative. [ABSTRACT FROM AUTHOR]

Sainfoin, Onobrychis viciifolia Scop., is an introduced forage legume grown on a limited basis in the Rocky Mountain region of the northern United States and southern Canada (Heath et al., 1973). The major attributes of sainfoin are its non-bloating characteristic as green forage and the high digestible nutrient content. It is drought tolerant, resistant to the alfalfa weevil, winter hardy, and produces good early spring growth tolerant to frost. If commercial Rhizobium inoculant is used, it requires no nitrogen fertilizer and has a low requirement for phosphorous. It has extremely good forage palatability. Therefore, it has the potential to provide a sustainable pasture system without required chemical inputs. The objective of our study was to determine if the combination of meadow bromegrass and our nematode-resistant sainfoin line would in crease the sustainability of sainfoin in the presence of the northern root-knot nematode (NRKN). Under environmentally controlled conditions, initial population density of five NRKN (Meloidogyne hapla Chitwood) eggs/cm3 soil resulted in 100% of sainfoin (Onobrychis viciifolia Scop.) roots with galls after70 days. One hundred percent of remaining sainfoin plants were galled in two other con trolled studies after 6 months, and 86% were galled in a field study after 2 years. Resistance to NRKN in an experimental sainfoin line was expressed in lower plant mortality, lower root galling, and lower egg production, compared to the susceptible cultivar Remont. The NRKN also appeared to be parasitic on roots of 'Regar' bromegrass (Bromus riparius Rehm.). Although root and shoot biomass were both reduced, there was no reproduction on roots and no bromegrass plants were killed. Under relatively high nematode soil populations, intercropping sainfoin with bromegrass provided no protection for sainfoin against the NRKN in either environmentally controlled or field plot studies. Our study further documents the extreme susceptibility [ABSTRACT FROM AUTHOR]

CAtiOn/H+ exchangers (CAxs) are integral membrane proteins that transport Ca2+ or other cations by exchange with protons. While several yeast and plant CAX proteins have been characterized, no functional analysis of a vertebrate CAX homologue has yet been reported. In this study, we further characterize a CAX from yeast, VNXl and initiate characterization ofa zebrafish CAX (Cax I). Localization studies indicated that both Vnxl and CaxI proteins are found in endomembrane compartments. Biochemical characterization of endomembrane fractions from vnx I mutant cells and zebrafish Cax I-expressing yeast cells suggested that both yeast and fish CAXs have Ca2+/H+ antiport activities. Additionally, the vnxl mutation was associated with heightened pH-sensitivity. In zebrafish embryos, cax I was specifically expressed in neural crest cells. Morpholino knockdown ofcaxl caused defects in neural crest development, including alterations in pigmentation, defects in jaw development, and reduction in expression of the neural crest marker, Pax7. Collectively, our findings provide insights into Vnxl function and support an unexpected role of CAX transporters in animal growth and development. [ABSTRACT FROM AUTHOR]

In plants, high capacity tonoplast cation/H+ antiport is mediated in part by a family of cation exchanger (CAX) transporters. Functional association between CAX1 and CAX3 has previously been shown. In this study we further examine the interactions between CAX protein domains through the use of nonfunctional halves of CAX transporters. We demonstrate that a protein coding for an N-terminal half of an activated variant ofCAX1 (sCAX1) can associate with the C-terminal half of either CAX1 or CAX3 to form a functional transporter that may exhibit unique transport properties. Using yeast split ubiquitin, inpianta bimolecular fluorescence complementation, and gel shift experiments, we demonstrate a physical interaction among the half proteins. Moreover, the half-proteins both independently localized to the same yeast endomembrane. Co-expressing variants of N- and C-terminal halves of CAX1 and CAX3 in yeast suggested that the N-terminal region mediates Ca2+ transport, whereas the C-terminal half defines salt tolerance phenotypes. Furthermore, in yeast assays, auto-inhibited CAX1 could be differentially activated by CAX split proteins. The N-terminal half of CAX1 when co-expressed with CAX1 activated Ca2+ transport, whereas co-expressing C-terminal halves of CAX variants with CAX1 conferred salt tolerance but no apparent Ca2+ transport. These findings demonstrate plasticity through hetero-CAX complex formation as well as a novel means to engineer CAX transport. [ABSTRACT FROM AUTHOR]

Regulation of Ca(2+)/H(+) antiporters may be an important function in determining the duration and amplitude of cytosolic Ca(2+) oscillations. Previously the Arabidopsis Ca(2+)/H(+) transporter, CAX1 (cation exchanger 1), was identified by its ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. Recently, a 36-amino acid N-terminal regulatory region on CAX1 has been identified that inhibits CAX1-mediated Ca(2+)/H(+) antiport. Here we show that a synthetic peptide designed against the CAX1 36 amino acids inhibited Ca(2+)/H(+) transport mediated by an N-terminal-truncated CAX1 but did not inhibit Ca(2+) transport by other Ca(2+)/H(+) antiporters. Ca(2+)/H(+) antiport activity measured from vacuolar-enriched membranes of Arabidopsis root was also inhibited by the CAX1 peptide. Through analyzing CAX chimeric constructs the region of interaction of the N-terminal regulatory region was mapped to include 7 amino acids (residues 56-62) within CAX1. The CAX1 N-terminal regulatory region was shown to physically interact with this 7-amino acid region by yeast two-hybrid analysis. Mutagenesis of amino acids within the N-terminal regulatory region implicated several residues as being essential for regulation. These findings describe a unique mode of antiporter autoinhibition and demonstrate the first detailed mechanisms for the regulation of a Ca(2+)/H(+) antiporter from any organism.