Loss of acidification of anterior prostate fluids inAtp12a-null mutant mice indicates that nongastric H-K-ATPase functions as proton pump in vivo

2006 ◽  
Vol 291 (2) ◽  
pp. C366-C374 ◽  
Author(s):  
Nikolay B. Pestov ◽  
Tatyana V. Korneenko ◽  
Mikhail I. Shakhparonov ◽  
Gary E. Shull ◽  
Nikolai N. Modyanov
Keyword(s):  
Proton Pump ◽  
Functional Expression ◽  
Α Subunit ◽  
Gene Ablation ◽  
Atpase Gene ◽  
Anterior Prostate ◽  
Null Mutant Mice ◽  
Apical Membranes ◽  
Prostate Epithelium

The physiological functions of nongastric (colonic) H-K-ATPase (gene symbol Atp12a), unlike those of Na-K-ATPase and gastric H-K-ATPase, are poorly understood. It has been suggested that it pumps Na+more efficiently than H+; however, so far, there is no direct evidence that it pumps H+in vivo. Previously, we found that the nongastric H-K-ATPase α-subunit is expressed in apical membranes of rodent anterior prostate epithelium, in a complex with the Na-K-ATPase β1-subunit. Here we report the effects of Atp12a gene ablation on polarization of the β1-subunit and secretory function of the anterior prostate. In nongastric H-K-ATPase-deficient prostate, the Na-K-ATPase α-subunit resided exclusively in basolateral membranes; however, the β1-subunit disappeared from apical membranes, demonstrating that β1is an authentic subunit of nongastric H-K-ATPase in vivo and that apical localization of β1in the prostate is completely dependent on its association with the nongastric H-K-ATPase α-subunit. A remarkable reduction in acidification of anterior prostate fluids was observed: pH 6.38 ± 0.14 for wild-type mice and 6.96 ± 0.10 for homozygous mutants. These results show that nongastric H-K-ATPase is required for acidification of luminal prostate fluids, thereby providing a strong in vivo correlate of previous functional expression studies demonstrating that it operates as a proton pump.

Dietary Na(+)-restriction prevents development of functional Na+ channels in taste cell apical membranes: proof by in vivo membrane voltage perturbation

1993 ◽  
Vol 70 (4) ◽  
pp. 1713-1716 ◽  
Author(s):  
Q. Ye ◽  
R. E. Stewart ◽  
G. L. Heck ◽  
D. L. Hill ◽  
J. A. DeSimone
Keyword(s):  
Voltage Clamp ◽  
Functional Expression ◽  
Voltage Sensitivity ◽  
Na Channels ◽  
Neural Responses ◽  
Positive Voltage ◽  
Taste Transduction ◽  
Contrast Ct ◽  
Apical Membranes

1. Chorda tympani (CT) neural responses to NaCl were recorded while the potential across the apical membrane of taste cells was perturbed by voltage clamp in rats fed a Na(+)-restricted diet pre- and postnatally (Na(+)-restricted rats) and in controls. 2. Control rats gave CT responses that were enhanced at negative voltage clamp and suppressed at positive voltage clamp. In contrast, CT responses from Na(+)-restricted rats were virtually voltage insensitive. 3. Analysis of the voltage-sensitivity of the CT response shows that Na(+)-restricted rats have < 10% of the density of functional apical Na+ channels normally present in control rats demonstrating that early dietary Na(+)-restriction prevents the functional expression of these key elements in salt taste transduction. Furthermore, the data demonstrate the value of this technique in assessing involvement of distinct cellular domains in taste transduction.

scholarly journals Functional expression of a GFP-tagged Kv1.5 α-subunit in mouse ventricle

2001 ◽  
Vol 281 (5) ◽  
pp. H1955-H1967 ◽  
Author(s):  
Huilin Li ◽  
Weinong Guo ◽  
Haodong Xu ◽  
Rebecca Hood ◽  
Andrew T. Benedict ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Functional Expression ◽  
Cardiac Cells ◽  
Cell Surface Expression ◽  
Delayed Rectifier ◽  
Wild Type ◽  
Α Subunit ◽  
Voltage Gated

The experiments here were undertaken to determine the feasibility of increasing the cell surface expression of voltage-gated ion channels in cardiac cells in vivo and to explore the functional consequences of ectopic channel expression. Transgenic mice expressing a green fluorescent protein (GFP)-tagged, voltage-gated K+(Kv) channel α-subunit, Kv1.5-GFP, driven by the cardiac-specific α-MHC promoter, were generated. In recent studies, Kv1.5 has been shown to encode the micromolar 4-aminopyridine (4-AP)-sensitive delayed rectifier K+ current ( I K,slow) in mouse myocardium. Unexpectedly, Kv1.5-GFP expression is heterogeneous in the ventricles of these animals. Although no electrocardiographic abnormalities were evident, expression of Kv1.5-GFP results in marked decreases in action potential durations in GFP-positive ventricular myocytes. In voltage-clamp recordings from GFP-positive ventricular myocytes, peak outward K+ currents are significantly higher, and their waveforms are distinct from those recorded from wild-type cells. Pharmacological experiments revealed a selective increase in a micromolar 4-AP-sensitive current, similar to the 4-AP-sensitive component of I K,slow in wild-type cells. The inactivation rate of the “overexpressed” current, however, is significantly slower than the Kv1.5-encoded component of I K,slow in wild-type cells, suggesting differences in association with accessory subunits and/or posttranslational processing.

Identification of the β-subunit for nongastric H-K-ATPase in rat anterior prostate

2004 ◽  
Vol 286 (6) ◽  
pp. C1229-C1237 ◽  
Author(s):  
Nikolay B. Pestov ◽  
Tatyana V. Korneenko ◽  
Rossen Radkov ◽  
Hao Zhao ◽  
Mikhail I. Shakhparonov ◽  
...  
Keyword(s):  
Structural Organization ◽  
Anterior Lobe ◽  
Β Subunit ◽  
Rt Pcr ◽  
Α Subunit ◽  
Direct Demonstration ◽  
Subunit Isoforms ◽  
Anterior Prostate ◽  
Apical Membranes

The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase α-subunit (αng) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to α1-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase β-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of β-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three β-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is β1 > β3 ≫ β2. Immunohistochemical experiments demonstrate that β1 is present predominantly in apical membranes, coinciding with αng, whereas β3 is localized in the basolateral compartment, coinciding with α1. This is the first direct demonstration of the αng-β1 colocalization in situ indicating that, in rat AP, αng associates only with β1. The existence of αng-β1 complex has been confirmed by immunoprecipitation experiments. These results indicate that β1-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

1999 ◽  
Vol 19 (12) ◽  
pp. 8422-8432 ◽  
Author(s):  
Olivier Donzé ◽  
Didier Picard
Keyword(s):  
Amino Acid ◽  
Constitutive Expression ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Restrictive Temperature ◽  
Α Subunit ◽  
Macbecin I

ABSTRACT The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

scholarly journals Interfering with VE-PTP stabilizes endothelial junctions in vivo via Tie-2 in the absence of VE-cadherin

2015 ◽  
Vol 212 (13) ◽  
pp. 2267-2287 ◽  
Author(s):  
Maike Frye ◽  
Martina Dierkes ◽  
Verena Küppers ◽  
Matthias Vockel ◽  
Janina Tomm ◽  
...  
Keyword(s):  
Tyrosine Phosphatase ◽  
Endothelial Barrier ◽  
Vascular Endothelial ◽  
Leukocyte Transmigration ◽  
Gene Ablation ◽  
Nonmuscle Myosin Ii ◽  
Endothelial Junctions ◽  
The Stability ◽  
Endothelial Junction

Vascular endothelial (VE)–protein tyrosine phosphatase (PTP) associates with VE-cadherin, thereby supporting its adhesive activity and endothelial junction integrity. VE-PTP also associates with Tie-2, dampening the tyrosine kinase activity of this receptor that can support stabilization of endothelial junctions. Here, we have analyzed how interference with VE-PTP affects the stability of endothelial junctions in vivo. Blocking VE-PTP by antibodies, a specific pharmacological inhibitor (AKB-9778), and gene ablation counteracted vascular leak induction by inflammatory mediators. In addition, leukocyte transmigration through the endothelial barrier was attenuated. Interference with Tie-2 expression in vivo reversed junction-stabilizing effects of AKB-9778 into junction-destabilizing effects. Furthermore, lack of Tie-2 was sufficient to weaken the vessel barrier. Mechanistically, inhibition of VE-PTP stabilized endothelial junctions via Tie-2, which triggered activation of Rap1, which then caused the dissolution of radial stress fibers via Rac1 and suppression of nonmuscle myosin II. Remarkably, VE-cadherin gene ablation did not abolish the junction-stabilizing effect of the VE-PTP inhibitor. Collectively, we conclude that inhibition of VE-PTP stabilizes challenged endothelial junctions in vivo via Tie-2 by a VE-cadherin–independent mechanism. In the absence of Tie-2, however, VE-PTP inhibition destabilizes endothelial barrier integrity in agreement with the VE-cadherin–supportive effect of VE-PTP.

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