Regulation of NHE3 activity by G protein subunits in renal brush-border membranes

2000 ◽  
Vol 278 (4) ◽  
pp. R1064-R1073 ◽  
Author(s):  
Frederick E. Albrecht ◽  
Jing Xu ◽  
Orson W. Moe ◽  
Ulrich Hopfer ◽  
William F. Simonds ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Brush Border ◽  
Second Messengers ◽  
Protein Subunit ◽  
Intact Cells ◽  
Protein Subunits ◽  
Brush Border Membranes ◽  
Renal Brush Border Membrane ◽  
Renal Brush Border

NHE3 activity is regulated by phosphorylation/dephosphorylation processes and membrane recycling in intact cells. However, the Na+/H+ exchanger (NHE) can also be regulated by G proteins independent of cytoplasmic second messengers, but the G protein subunits involved in this regulation are not known. Therefore, we studied G protein subunit regulation of NHE3 activity in renal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmic components and second messengers. Basal NHE3 activity was not regulated by Gsα or Giα, because antibodies to these G proteins by themselves were without effect. The inhibitory effect of D1-like agonists on NHE3 activity was mediated, in part, by Gsα, because it was partially reversed by anti-Gsα antibodies. Moreover, the amount of Gsα that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-border membranes and renal proximal tubule cells. Furthermore, guanosine 5′- O-(3-thiotriphosphate) but not guanosine 5′- O-(2-thiodiphosphate), the inactive analog of GDP, increased the amount of Gsα that coimmunoprecipitated with NHE3. The α2-adrenergic agonist, UK-14304 or pertussis toxin (PTX) alone had no effect on NHE3 activity, but UK-14304 and PTX treatment attenuated the D1-like receptor-mediated NHE3 inhibition. The ability of UK-14304 to attenuate the D1-like agonist effect was not due to Giα, because the attenuation was not blocked by anti-Giα antibodies or by PTX. Anti-Gβcommon antibodies, by themselves, slightly inhibited NHE3 activity but had little effect on D1-like receptor-mediated NHE3 inhibition. However, anti-Gβcommon antibodies reversed the effects of UK-14304 and PTX on D1-like agonist-mediated NHE3 inhibition. These studies provide concrete evidence of a direct regulatory role for Gsα, independent of second messengers, in the D1-like-mediated inhibition of NHE3 activity in rat renal BBMV. In addition, β/γ dimers of heterotrimeric G proteins appear to have a stimulatory effect on NHE3 activity in BBMV.

Ion channel regulation by G proteins

1995 ◽  
Vol 75 (4) ◽  
pp. 865-885 ◽  
Author(s):  
K. Wickman ◽  
D. E. Clapham
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
G Protein ◽  
G Proteins ◽  
Second Messengers ◽  
Channel Activity ◽  
Ion Channel Regulation ◽  
Protein Subunits ◽  
Channel Regulation ◽  
Intracellular Cascades

Ion channels are poised uniquely to initiate, mediate, or regulate such distinct cellular activities as action potential propagation, secretion, and gene transcription. In retrospect, it is not surprising that studies of ion channels have revealed considerable diversities in their primary structures, regulation, and expression. From a functional standpoint, the various mechanisms coopted by cells to regulate channel activity are particularly fascinating. Extracellular ligands, membrane potential, phosphorylation, ions themselves, and diffusible second messengers are all well-established regulators of ion channel activity. Heterotrimeric GTP-binding proteins (G proteins) mediate many of these types of ion channel regulation by stimulating or inhibiting phosphorylation pathways, initiating intracellular cascades leading to elevation of cytosolic Ca2+ or adenosine 3',5'-cyclic monophosphate levels, or by generating various lipid-derived compounds. In some cases, it seems that activated G protein subunits can interact directly with ion channels to elicit regulation. Although there is currently little direct biochemical evidence to support such a mechanism, it is the working hypothesis for the most-studied G protein-regulated ion channels.

Gβ regulation of Na/H exchanger-3 activity in rat renal proximal tubules during development

2000 ◽  
Vol 278 (4) ◽  
pp. R931-R936 ◽  
Author(s):  
Xiao Xi Li ◽  
Frederick E. Albrecht ◽  
Jean E. Robillard ◽  
Gilbert M. Eisner ◽  
Pedro A. Jose
Keyword(s):  
G Protein ◽  
Second Messengers ◽  
Membrane Vesicles ◽  
Inhibitory Effect ◽  
Protein Subunit ◽  
Adult Rats ◽  
Renal Brush Border Membrane ◽  
Sodium Hydrogen Exchanger ◽  
Old Rats ◽  
Renal Tubular

The decreased natriuretic action of dopamine in the young has been attributed to decreased generation of cAMP by the activated renal D1-like receptor. However, sodium/hydrogen exchanger (NHE) 3 activity in renal brush-border membrane vesicles (BBMV) can be modulated independent of cytoplasmic second messengers. We therefore studied D1-like receptor regulation of NHE activity in BBMVs in 2-, 4-, and 12-wk-old (adult) rats. Basal NHE activity was least in 2-wk-old compared with 4- and 12-wk-old rats. D1-like agonist (SKF-81297) inhibition of NHE activity was also least in 2-wk-old (−1 ± 9%, n = 3) compared with 4 (−15 ± 5%, n = 6)- and 12 (−65 ± 4%, n = 6)-wk-old rats. The decreased response to the D1-like agonist in BBMV was not caused by decreased D1 receptors or NHE3 expression in the young. Gsα, which inhibits NHE3 activity by itself, coimmunoprecipitated with NHE3 to the same extent in 2-wk-old and adult rats. Gsα function was also not impaired in the young because guanosine 5′- O-(3-thiotriphosphate) decreased NHE activity to a similar extent in 4-wk-old and adult rats. Gαi-3 protein expression in BBMV also did not change with age. In contrast, Gβ expression and the amount of Gβ that coimmunoprecipitated with NHE3 in BBMV was greatest in 2-wk-old rats and decreased with age. Gβ common antibodies did not affect D1-like agonist inhibition of NHE activity in adult rats (8%) but markedly increased it (48%)in 4-wk-old rats. We conclude that the decreased inhibitory effect of D1-like receptors on NHE activity in BBMV in young rats is caused, in part, by the increased expression and activity of the G protein subunit Gβ/γ. The direct regulation of NHE activity by G protein subunits may be an important step in the maturation of renal tubular ion transport.

Transport of urate and p-aminohippurate in rabbit renal brush-border membranes

1990 ◽  
Vol 258 (5) ◽  
pp. F1145-F1153 ◽  
Author(s):  
F. Martinez ◽  
M. Manganel ◽  
C. Montrose-Rafizadeh ◽  
D. Werner ◽  
F. Roch-Ramel
Keyword(s):  
Brush Border ◽  
Transport Mechanism ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Second Step ◽  
Filtration Method ◽  
Brush Border Membranes ◽  
Renal Brush Border Membrane ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

The mechanisms involved in urate and p-aminohippurate (PAH) transport in the rabbit renal brush-border membrane were investigated through study of membrane vesicles. Transport of [14C]urate and [3H]PAH was measured by a rapid filtration method. As previously reported by others, no OH(-)-PAH exchanger could be demonstrated by imposing an outwardly directed OH- gradient (pHin 7.4, pHout 6). In contrast, an OH(-)-lactate exchanger (or H(+)-lactate cotransport) was demonstrated. In the presence of valinomycin and an inwardly directed K+ gradient, both [14C]urate and [3H]PAH vesicle uptake were stimulated, demonstrating a potential-driven transport of these two anions. Probenecid, PAH, or cold urate decreased potential-driven urate uptake, suggesting that this transport was facilitated by a specific transport mechanism. The potential-driven urate transport described here may play a role in the second step of urate secretion in rabbits, because rate (or PAH) is transported across the brush-border membrane from the negative interior of the cell to the more positive omen.

scholarly journals Electrogenicity of phosphate transport by renal brush-border membranes

1988 ◽  
Vol 252 (3) ◽  
pp. 801-806 ◽  
Author(s):  
R Béliveau ◽  
H Ibnoul-Khatib
Keyword(s):  
Membrane Potential ◽  
Brush Border ◽  
Phosphate Uptake ◽  
Electrical Potential ◽  
Membrane Potentials ◽  
Experimental Conditions ◽  
Brush Border Membranes ◽  
Renal Brush Border Membrane ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.

Low-affinity transport of pyroglutamyl-histidine in renal brush-border membrane vesicles

1989 ◽  
Vol 257 (5) ◽  
pp. C971-C975 ◽  
Author(s):  
H. A. Skopicki ◽  
K. Fisher ◽  
D. Zikos ◽  
G. Flouret ◽  
D. R. Peterson
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Luminal Membrane ◽  
Renal Brush Border Membrane ◽  
Proximal Tubular ◽  
Renal Brush Border

These studies were performed to determine if a low-affinity carrier is present in the luminal membrane of proximal tubular cells for the transport of the dipeptide, pyroglutamyl-histidine (pGlu-His). We have previously described the existence of a specific, high-affinity, low-capacity [transport constant (Kt) = 9.3 X 10(-8) M, Vmax = 6.1 X 10(-12) mol.mg-1.min-1] carrier for pGlu-His in renal brush-border membrane vesicles. In the present study, we sought to demonstrate that multiple carriers exist for the transport of a single dipeptide by determining whether a low-affinity carrier also exists for the uptake of pGlu-His. Transport of pGlu-His into brush-border membrane vesicles was saturable over the concentration range of 10(-5)-10(-3) M, yielding a Kt of 6.3 X 10(-5) M and a Vmax of 2.2 X 10(-10) mol.mg-1.min-1. Uptake was inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and carnosine but not by the tripeptide pyroglutamyl-histidyl-prolinamide. We conclude that 1) pGlu-His is transported across the luminal membrane of the proximal tubule by multiple carriers and 2) the lower affinity carrier, unlike the higher affinity carrier, is nonspecific with respect to other dipeptides.

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