Reconstitution and partial purification of several Na+ cotransport systems from renal brush-border membranes. Properties of the L-glutamate transporter in proteoliposomes.

Koepsell, Hermann; Korn, Klaus; Ferguson, Donald; Menuhr, Helmut; Ollig, Doris; Haase, Winfried

doi:10.1016/s0021-9258(20)82176-0

“…To measure organic cation uptake by transporters rOct1 and rOct2 that are driven by substrate gradient and membrane potential ,, and uptake of PAH – by rOat1 that was only observed in the presence of a trans- KG 2− gradient ,, , we reconstituted the transporters into large cholesterol-rich proteoliposomes with diameters between 0.5 and 2 µm , . The employed reconstitution protocol had been optimized to analyze transport activities of electrogenic Na + cotransporters , .…”

Section: Discussionmentioning

confidence: 99%

“…To measure organic cation uptake by transporters rOct1 and rOct2 that are driven by substrate gradient and membrane potential ,, and uptake of PAH – by rOat1 that was only observed in the presence of a trans- KG 2− gradient ,, , we reconstituted the transporters into large cholesterol-rich proteoliposomes with diameters between 0.5 and 2 µm , . The employed reconstitution protocol had been optimized to analyze transport activities of electrogenic Na + cotransporters , . Because of their relatively large size and low passive permeability, these proteoliposomes are well suited to measurement of initial uptake rates of transporters that are driven by ion gradients, i.e., cotransporters or antiporters.…”

Section: Discussionmentioning

confidence: 99%

Cell Free Expression and Functional Reconstitution of Eukaryotic Drug Transporters

Keller

¹

,

Schwarz

²

,

Bernhard

³

et al. 2008

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Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy- sn-glycero-3-[phospho- rac-(1-glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1 mL reaction vessels 0.13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0.5% CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis-Menten K m values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH (-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH (-)/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH (-) uptake was observed only with alpha-ketoglutarate (KG (2-)) on the trans side, and trans-KG (2-) increased the PAH (-) concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V max of PAH (-)/KG (2-) antiport was increased by Cl (-) in a manner independent of gradients, and PAH (-)/KG (2-) antiport was independent of membrane potential in the absence or presence of Cl (-).

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“…13,14 Similarly, membrane vesicles prepared from the brush border of the intestine [15][16][17] and of kidney tubules were shown to take up glutamate with high affinity. [18][19][20][21][22][23][24] The 2 first glutamate transporters (or excitatory amino acid transporters), EAAT1 (GLAST; Slc1a3) and EAAT2 (GLT-1; Slc1a2), were cloned after the proteins had been isolated from rat brain. [25][26][27] On the other hand, EAAT3 (EAAC1; Slc1a1) was identified by expression cloning from rabbit jejunum.…”

Section: Introductionmentioning

confidence: 99%

Expression of Glutamate Transporters in Mouse Liver, Kidney, and Intestine

Hu

¹

,

Ottestad-Hansen

²

,

Holmseth

³

et al. 2018

J Histochem Cytochem.

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Glutamate transport activities have been identified not only in the brain, but also in the liver, kidney, and intestine. Although glutamate transporter distributions in the central nervous system are fairly well known, there are still uncertainties with respect to the distribution of these transporters in peripheral organs. Quantitative information is mostly lacking, and few of the studies have included genetically modified animals as specificity controls. The present study provides validated qualitative and semi-quantitative data on the excitatory amino acid transporter (EAAT)1-3 subtypes in the mouse liver, kidney, and intestine. In agreement with the current view, we found high EAAT3 protein levels in the brush borders of both the distal small intestine and the renal proximal tubules. Neither EAAT1 nor EAAT2 was detected at significant levels in murine kidney or intestine. In contrast, the liver only expressed EAAT2 (but 2 C-terminal splice variants). EAAT2 was detected in the plasma membranes of perivenous hepatocytes. These cells also expressed glutamine synthetase. Conditional deletion of hepatic EAAT2 did neither lead to overt neurological disturbances nor development of fatty liver.

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Principles of Electrolyte Transport Across Plasma Membranes of Renal Tubular Cells

Burckhardt

¹

,

Greger

²

1992

Comprehensive Physiology

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The sections in this article are: Principles of Sodium Reabsorption Coupling of Sodium Transport to Bicarbonate Absorption Coupling of Sodium Reabsorption to Chloride Rheogenic Sodium Reabsorption Comparative Energetic Aspects Principles of Proton Secretion Sodium‐Dependent Proton Secretion Sodium‐Independent Proton Secretion Comparative Energetic Aspects Principles of Sodium‐Coupled Solute Absorption Specificity Source of Energy Homocellular Regulation Mechanisms Principles of Solute Uptake Across the Basolateral Membrane Anion Transport Systems in the Basolateral Membrane PAH Secretion: A Tertiary Active Process Conclusion

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Reconstitution and partial purification of several Na+ cotransport systems from renal brush-border membranes. Properties of the L-glutamate transporter in proteoliposomes.

Cited by 63 publications

References 39 publications

Cell Free Expression and Functional Reconstitution of Eukaryotic Drug Transporters

Cell Free Expression and Functional Reconstitution of Eukaryotic Drug Transporters

Expression of Glutamate Transporters in Mouse Liver, Kidney, and Intestine

Principles of Electrolyte Transport Across Plasma Membranes of Renal Tubular Cells

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