Role of the trace amine associated receptor 5 (TAAR5) in the sensorimotor functions.

Classical monoamines are well-known modulators of sensorimotor neural networks. However, the role of trace amines and their receptors in sensorimotor function remains unexplored. Using trace amine-associated receptor 5 knockout (TAAR5-KO) mice, that express beta-galactosidase mapping its localization, we observed TAAR5 expression in the Purkinje cells of the cerebellum and the medial vestibular nucleus, suggesting that TAAR5 might be involved in the vestibular and motor control. Accordingly, in various behavioral tests, TAAR5-KO mice demonstrated lower endurance, but better coordination and balance compared to wild-type controls. Furthermore, we found specific changes in striatal local field potentials and motor cortex electrocorticogram, such as a decrease in delta and an increase in theta oscillations of power spectra, respectively. The obtained data indicate that TAAR5 plays a considerable role in regulation postural stability, muscle force, balance, and motor coordination during active movements, likely via modulation of monoaminergic systems at different levels of sensorimotor control involving critical brain areas such as the brainstem, cerebellum, and forebrain.

[The Familial Hypercholesterolemia Caused by a Novel Human Low Density Lipoprotein Receptor Gene Mutation c.1327 T>C (p.W433R)].

During investigation of molecular nature of familial hypercholesterolemia (FH) in Petrozavodsk (Russia) cohort of patients a novel low density lipoprotein (LDL) receptor gene mutation was found. This mutation designated c.1327 T>C (W443R [W422R]) was predicted to cause substitution of arginine for tryptophan residue in the very conservative -propeller domain of the LDL receptor. Inheritance of the new mutation was traced in four generations and its cosegregation with hypercholesterolemia phenotype was observed. Despite the predicted pathogenic effect of the mutation, ischemic heart disease in the pedigree was mild or absent. We consider identification of this mutation in the pedigree extremely helpful to start preventive medical treatment in affected patients.

Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles.

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles.

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCß3, E-cadherin, p120, ß-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

Half-lives of plasma membrane Na(+)/H(+) exchangers NHE1-3: plasma membrane NHE2 has a rapid rate of degradation.

The Na(+)/H(+) exchangers NHE2 and NHE3 are involved in epithelial Na(+) and HCO absorption. To increase insights into the functions of NHE2 vs. NHE3, we compared their cellular processing with each other and with the housekeeping isoform NHE1. Using biotinylated exchanger, we determined that the half-life of plasma membrane NHE2 was short (3 h) compared with that of NHE1 (24 h) and NHE3 (14 h) in both PS120 fibroblasts and Caco-2 cells. NHE2 transport and plasma membrane levels were reduced by 3 h of Brefeldin A treatment, whereas NHE1 was unaffected. NHE2 was degraded by the lysosomes but not proteosomes, as demonstrated by increasing levels of endocytosed NHE2 protein after inhibition of the lysosomes, but not with proteosome inhibition. Unlike that of NHE3, basal NHE2 transport activity was not affected by phosphatidylinositol 3-kinase inhibition and did not appear to be localized in the juxtanuclear recycling endosome. Therefore, for NHE2, protein degradation and/or protein synthesis probably play important roles in its basal and regulated states. These results suggest fundamental differences in the cellular processing and trafficking of NHE2 and NHE3. These differences may underlie the specialized roles that these exchangers play in epithelial cells.

Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger.

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells. Site-specific mutagenesis was used to examine the functional role of conserved, polar amino-acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino-acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of Glu391 resulted in only a partial reduction in activity. The Glu262-->Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutated Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H+ exchanger activity. The Glu262-->Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results support the hypothesis that side-chain oxygen atoms in a few, critically placed amino acids are important in Na+/H+ exchanger activity and the acidic amino-acid residues at positions 262, 267 and 391 are good candidates for being involved in Na+ coordination by the protein.

Characterization of a histidine rich cluster of amino acids in the cytoplasmic domain of the Na+/H+ exchanger.

We examined the function of a highly conserved Histidine rich sequence of amino acids found in the carboxyl-terminal of the Na+/H+ exchanger (NHE1). A fusion protein containing the sequence HYGHHH (540545) and the balance of the carboxyl terminal of the protein did not bind calcium but bound to an immobilized metal affinity column and could be used to partially purify the exchanger protein. Mutation of the sequence to either HYGAAA or HYGRRR did not affect activity of the intact protein. Mutation to HHHHHH did not affect proton activation of the Na+/H+ exchanger or localization but caused a decreased maximal velocity suggesting that this conserved sequence is important in maximal activity of the Na+/H+ exchanger.

Calcium and osmotic regulation of the Na+/H+ exchanger in neonatal ventricular myocytes.

Intracellular pH regulation in primary cultures of neonatal cardiac myocytes has been characterized. Myocytes were exposed to hyperosmolar solutions to examine the effects on pH regulation by the Na+/H+ exchanger. Exposure to 100 mM NaCl, sorbitol, N-methyl-D-glucamine, or choline chloride all caused significant increases in steady state pHi in myocytes. Omission of extracellular calcium or administration of calmodulin antagonists reduced the osmotic activation of the exchanger. The myosin light-chain inhibitor ML-7 completely blocked osmotic activation of the exchanger suggesting that myosin light-chain kinase is involved in osmotic activation of the exchanger in the myocardium. The calmodulin-dependent protein kinase II inhibitor KN-93 inhibited the rate of recovery from an acute acid load as did trifluoperazine (TFP) and the calmodulin blocker W7, [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. Addition of the calcium ionophore ionomycin caused a large increase in resting pHi in isolated myocytes. However, this effect was largely resistant to HMA (5-(N,N-hexamethylene)-amiloride) indicating that an alternative mechanism of pHi regulation is responsible. The results demonstrate that the Na+/H+ exchanger of the neonatal myocardium is responsive to calcium and osmotically responsive pathways and that myosin light-chain kinase is a key protein involved in mediating the osmotic response.

Regulation and characterization of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous protein present in all mammalian cell types that functions to remove one intracellular H+ for one extracellular Na+. Several isoforms of the protein exist, which are referred to as NHE1 to NHE6 (for Na+/H+ exchanger one through six). The NHE1 protein was the first isoform cloned and studied in a variety of systems. This review summarizes recent papers on this protein, particularly those that have examined regulation of the protein and its expression and activity.

The Na+/e- stoichiometry of the Na+-motive NADH:quinone oxidoreductase in Vibrio alginolyticus.

A method is proposed to estimate the stoichiometries of primary Na+-pumps in intact bacterial cells. It is based on technique when the H+/e- stoichiometry is measured in the presence of protophorous uncoupler and in the absence of penetrating ions other than H+. Under these conditions, the H+ influx discharges membrane potential generated by the Na+ pump so the Na+/e- and H+/e- ratios become equal. Using this approach it is shown that the Na+/e- ratio for the Na+-motive NADH:quinone oxidoreductase of Vibrio alginolyticus is equal to 0.71 +/- 0.06. The Na+/e- stoichiometry appears to be approximately 1, provided that the contribution of the non-coupled NADH:quinone oxidoreductase, which is resistant to low HQNO concentrations, is taken into account.

Aeration-dependent changes in composition of the quinone pool in Escherichia coli. Evidence of post-transcriptional regulation of the quinone biosynthesis.

The aeration-dependent changes in content of various quinones in Escherichia coli were found to be unaffected by a prokaryotic translation inhibitor chloramphenicol. In addition, this process was shown to be completely intact in cells with mutated fnr, arc and appY loci. It is assumed that E. coli possesses a special system of oxygen-dependent post-transcriptional regulation of the quinone biosynthetic pathways.

H+/e- stoichiometry for NADH dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli cells.

Anaerobically grown Escherichia coli cells were shown to acidify the reaction medium in response to oxygen or dimethyl sulfoxide (DMSO) pulses, with the H+/e- stoichiometry being close to 2.5 and 1.5, respectively. In the presence of the NADH dehydrogenase I (NDH-I) inhibitor 8-methyl-N-vanillyl-6-nonenamide (capsaicin) or in mutants lacking NDH-I, this ratio decreased to 1 for O2 and to 0 for DMSO. These data suggest that (i) the H+/e- stoichiometry for E. coli NDH-I is at least 1.5 and (ii) the DMSO reductase does not generate a proton motive force.

Cytochrome d induction in Escherichia coli growing under unfavorable conditions.

Growth of E. coli in the presence of the protonophorous uncoupler pentachlorophenol is shown to strongly enhance levels of cytochrome d, a putative Na(+)-motive oxidase. This effect was found to be arrested by chloramphenicol and stimulated by high Na+ concentration in the growth medium. The induction of cytochrome d takes place in a mutant deficient in the F0F1 ATP-synthase but does not occur in mutants deficient in either of two different components of the Arc system. Similar relationships were revealed when pentachlorophenol was replaced by ferricyanide and phenazine methosulfate, agents oxidizing the respiratory chain. Induction of cytochrome d is also shown to occur in riboflavin-deficient mutants growing in the presence of such low riboflavin concentrations as to be insufficient to maintain a high respiration rate. It is suggested (i) that it is delta mu H+ decrease rather than reduction of the respiratory chain that is the signal for the induction of cytochrome d, and (ii) the Arc system is involved in this type of metabolic regulation.