Na-dependent L-glutamate transport by eel intestinal BBMV: role of K+ and Cl-

1989 ◽  
Vol 257 (1) ◽  
pp. R180-R188
Author(s):  
P. M. Romano ◽  
G. A. Ahearn ◽  
C. Storelli
Keyword(s):  
Amino Acid ◽  
Glutamate Uptake ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Anguilla Anguilla ◽  
Competitive Inhibitor ◽  
Glutamate Transport ◽  
Electrical Potential Difference ◽  
Intestinal Brush Border Membrane ◽  
Transmembrane Electrical Potential

L-[3H]glutamate uptake into eel (Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) was a sigmoidal function of extravesicular Na, suggesting that two or more cations accompanied the amino acid during transport. L-[3H]glutamate influx illustrated the following kinetic constants: apparent membrane binding affinity (Kapp) = 0.80 +/- 0.12 mM; influx velocity (Jmax) = 2.61 +/- 0.31 nmol.mg protein-1.min-1; and permeability coefficient (P) = 0.65 +/- 0.10 microliters.mg protein-1. min-1. Results from the imposition of diffusion potentials across vesicle membranes using K-valinomycin or H-carbonyl-cyanide p-chloromethoxyphenylhydrazone suggested that Na-dependent L-glutamate transport was sensitive to transmembrane electrical potential difference. Extravesicular aspartate was a competitive inhibitor of L-[3H]glutamate influx [inhibitory constant (Ki) = 0.28 +/- 0.04 mM]. Intravesicular K and extravesicular Cl ions enhanced maximal amino acid influx and transient L-glutamate accumulation against a concentration gradient (overshoot). Intravesicular K reduced the Kapp of the membrane to L-glutamate, whereas extravesicular Cl increased L-glutamate Jmax. A model for L-[3H]glutamate transport is suggested involving the cotransport of at least two Na and one L-glutamate that is activated by one intravesicular K ion and at least two extravesicular Cl ions.

Sodium and calcium share the electrogenic 2 Na(+)-1 H+ antiporter in crustacean antennal glands

1990 ◽  
Vol 259 (5) ◽  
pp. F758-F767
Author(s):  
G. A. Ahearn ◽  
P. Franco
Keyword(s):  
Driving Forces ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Homarus Americanus ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Affinity Constant ◽  
Electrical Potential Difference ◽  
Static Head ◽  
Transmembrane Electrical Potential

Na uptake by short-circuited epithelial brush-border membrane vesicles of Atlantic lobster (Homarus americanus) antennal gland labyrinth was Cl independent, amiloride sensitive, and stimulated by a transmembrane H+ gradient [( H]i greater than [H]o; i is internal, o is external). Na influx (2.5-s uptake) was a sigmoidal function of [Na]o (25-400 mM) when pHi = 5.0 and pHo = 8.0 and followed the Hill equation for binding cooperatively [apparent maximal influx (Jmax) = 271 nmol.mg protein-1.s-1, apparent affinity constant for Na (KNa) = 310 mM Na, and Hill coefficient (n) = 2.41]. Amiloride acted as a competitive inhibitor of Na binding to two external sites with markedly dissimilar apparent amiloride affinities (Ki1 = 14 microM; Ki2 = 1,340 mM). Electrogenic Na-H antiport by these vesicles was demonstrated by equilibrium-shift experiments in which an imposed transmembrane electrical potential difference was the only driving force for exchange. A transport stoichiometry of 2 Na to 1 H was demonstrated with the static-head technique in which a balance of driving forces was attained with 10:1 Na gradient and 100:1 H gradient. External Ca, like amiloride, was a strong competitive inhibitor of Na-H exchange, acting at two sites on the outer vesicular face with markedly different apparent divalent cation affinities (Ki1 = 20 microM; Ki2 = 500 microM). Ca-H exchange by electrogenic Na-H antiporter was demonstrated in complete absence of Na by use of an outward H gradient in presence and absence of amiloride. Both external amiloride (Ki1 = 70 microM; Ki2 = 500 microM) and Na (Ki1 = 12 mM; Ki2 = 380 mM) were competitive inhibitors of Ca-H exchange. These results suggest that the electrogenic 2 Na-1 H exchanger characterized for this crustacean epithelium may also have a role in organismic Ca balance.

scholarly journals The K+-driven Amino Acid Cotransporter of the Larval Midgut of Lepidoptera: Is Na+ an Alternative Substrate

1992 ◽  
Vol 162 (1) ◽  
pp. 281-294
Author(s):  
G. M. HANOZET ◽  
V. F. SACCHI ◽  
S. NEDERGAARD ◽  
P. BONFANTI ◽  
S. MAGAGNIN ◽  
...  
Keyword(s):  
Amino Acid ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Electrical Potential Difference ◽  
Glycine Uptake ◽  
Larval Midgut ◽  
Control Value ◽  
Fixed Concentration ◽  
K Concentration ◽  
Transmembrane Electrical Potential

Amino acid accumulation within brush-border membrane vesicles (BBMV) from the larval midgut of Lepidoptera is driven by a K+ gradient. However, it can also be driven by a Na+ gradient, although with reduced efficiency. To examine the possibility that sodium and potassium ions are handled by the same amino acid transporter, glycine uptake into BBMV from Philosamia cynthia Drury was measured in the presence of a pH gradient and of a transmembrane electrical potential difference, i.e. in simulated ‘physiological’ conditions. The kinetics of glycine uptake at extravesicular saturating Na+ or K+ concentrations discloses a higher affinity of the cotransporter for the amino acid in the presence of Na+ but a maximum transport rate with K+. Glycine uptake at a fixed concentration as a function of external Na+ or K+ concentration yields curves that show saturation but do not fit a rectangular hyperbola, with Hill coefficients less than 1 with Na+ and greater than 1 with K+. These coefficients vary according to glycine concentration. Increasing the concentration of extravesicular Na+ at a saturating external K+ concentration reduced glycine uptake to 70% of the control value. This inhibition curve is compatible with competition between the two cations for the same cotransporter and with the presence of different kinetic constants with Na+ or K+. The data are consistent with a steady-state random two-substrate mechanism for glycine transport, with Na+ and K+ as alternative substrates.

K+-neutral amino acid symport of Bombyx mori larval midgut: a system operative in extreme conditions

1998 ◽  
Vol 274 (5) ◽  
pp. R1361-R1371 ◽  
Author(s):  
B. Giordana ◽  
M. G. Leonardi ◽  
M. Casartelli ◽  
P. Consonni ◽  
P. Parenti
Keyword(s):  
Amino Acid ◽  
Bombyx Mori ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Ph Gradient ◽  
Electrical Potential Difference ◽  
Posterior Midgut ◽  
Solution Bathing

The K+-dependent symporter for leucine and other neutral amino acids expressed along the midgut of the silkworm Bombyx mori operates with best efficiency in the presence of a steep pH gradient across the brush-border membrane, with external alkaline pH values up to 11, and an electrical potential difference (Δψ) of ∼200 mV. Careful determinations of leucine kinetics as a function of external amino acid concentrations between 50 and 1,000 μM, performed with brush-border membrane vesicles (BBMV) obtained from the middle and posterior midgut regions, revealed that the kinetic parameter affected by the presence of a ΔpH was the maximal rate of transport. The addition of Δψ caused a further marked increase of the translocation rate. At nonsaturating leucine concentrations in the solution bathing the external side of the brush-border membrane, leucine accumulation within BBMV and midgut cells was not only driven by the gradient of the driver cation K+ and Δψ but occurred also in the absence of K+. The ability of the symporter to translocate the substrate in its binary form allows the intracellular accumulation of leucine in the absence of K+, provided that a pH gradient, with alkaline outside, is present. The mechanisms involved in this accumulation are discussed.

An electroneutral anion exchange mechanism is present in brush-border membranes isolated from eel kidney

1997 ◽  
Vol 272 (4) ◽  
pp. R1143-R1148 ◽  
Author(s):  
S. Vilella ◽  
L. Ingrosso ◽  
V. Zonno ◽  
T. Schettino ◽  
C. Storelli
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Potential Gradient ◽  
Membrane Vesicles ◽  
Anguilla Anguilla ◽  
Brush Border Membrane Vesicles ◽  
Disulfonic Acid ◽  
Luminal Membrane ◽  
Transmembrane Electrical Potential

The mechanism of bicarbonate translocation across the luminal membrane of the eel (Anguilla anguilla) kidney tubular cells was studied by monitoring the uptake of H14CO3- into isolated brush-border membrane vesicles. Results indicate that the presence of a transmembrane outwardly directed Cl- gradient was able to transiently accumulate H14CO3- into the vesicular space, whereas neither an inwardly directed sodium gradient nor a transmembrane electrical potential gradient (inside positive) was able to stimulate the H14CO3- influx. This anion-dependent H14CO3- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that an anion exchanger was present in the brush-border membrane vesicles.

Na-dependent L-proline transport by eel intestinal brush-border membrane vesicles

1988 ◽  
Vol 255 (4) ◽  
pp. R648-R653 ◽  
Author(s):  
S. Vilella ◽  
G. A. Ahearn ◽  
G. Cassano ◽  
C. Storelli
Keyword(s):  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Anguilla Anguilla ◽  
Brush Border Membrane Vesicles ◽  
European Eel ◽  
Intestinal Brush Border Membrane ◽  
Diffusional Permeability ◽  
Wide Range

L-[3H]proline uptake by brush-border membrane vesicles prepared from intestinal mucosa of the European eel, Anguilla anguilla, was stimulated by a transmembrane Na gradient (out greater than in). Kinetic analysis of L-proline influx, under short-circuited membrane potential conditions, indicated the presence of an apparent single Na-dependent carrier process (Kapp = 0.23 +/- 0.04 mM and Jmax = 7.96 +/- 0.87 nmol.mg protein-1.min-1) and a nonsaturable transfer component with an apparent diffusional permeability (P) of 1.53 +/- 0.35 microliter.mg protein-1.min-1. An imposed transmembrane potential (inside negative) increased apparent L-proline binding affinity (lowered Kapp) without appreciably altering maximal amino acid influx (Jmax). Hill analysis of L-proline influx over a wide range of external Na concentrations indicated a 1:1 stoichiometry for Na-proline cotransport. Use of amino acid inhibitors of L-proline influx suggested that L-proline transfer may occur by either a classical Na-dependent A System with a wide substrate specificity or by the combination of Na-dependent PHE (phenylalanine preferring) and IMINO (proline, alpha-methylaminoisobutyric acid preferring) Systems.

scholarly journals Evidence for a low-affinity, high-capacity uniport for amino acids in Bombyx mori larval midgut

1998 ◽  
Vol 274 (5) ◽  
pp. R1372-R1375 ◽  
Author(s):  
M. G. Leonardi ◽  
M. Casartelli ◽  
P. Parenti ◽  
B. Giordana
Keyword(s):  
Amino Acids ◽  
Kinetic Analysis ◽  
Bombyx Mori ◽  
Electrical Potential ◽  
High Capacity ◽  
Membrane Vesicles ◽  
Electrical Potential Difference ◽  
Larval Midgut ◽  
Posterior Midgut ◽  
Transmembrane Electrical Potential

We investigated the kinetics of leucine influx as a funtion of external substrate concentration between 0.03 and 16 mM in brush-border membrane vesicles (BBMV) prepared from the middle region of Bombyx mori larval midgut. A detailed kinetic analysis of leucine uptake led to the identification, in parallel with the K+-dependent symporter for neutral amino acids, of a K+-independent, low-affinity, high-capacity system. The parameter values of the Michaelis constant (7.12 mM) and maximal rate of transport (4.48 nmol ⋅ 7 s−1 ⋅ mg protein−1) were not influenced by an external alkaline pH nor by a transmembrane electrical potential difference. The uniporter is poorly specific, as it displayed the following rank of preference: Leu, His, Val, Ile, Phe, Ser > Lys, Arg, Gln > Pro, 2-amino-2-norbornane-carboxylic acid, Ala, Gly. The kinetic analysis performed in BBMV prepared from the posterior midgut portion indicates that the low-affinity, high-capacity uniporter is present along the entire length of the silkworm larval midgut with similar expression and functional properties.

An anion exchanger mediates bile acid transport across the placental microvillous membrane

1993 ◽  
Vol 264 (6) ◽  
pp. G1016-G1023 ◽  
Author(s):  
R. Dumaswala ◽  
K. D. Setchell ◽  
M. S. Moyer ◽  
F. J. Suchy
Keyword(s):  
Bile Acids ◽  
Initial Rate ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Electrical Potential Difference ◽  
Maximal Velocity ◽  
Temperature Dependent ◽  
Transmembrane Electrical Potential ◽  
Microvillous Membrane Vesicles

Microvillous membrane vesicles from the term human placental syncytiotrophoblast were used to characterize further the properties of a transport mechanism for bile acids. Taurocholate (TC) uptake into an osmotically reactive intravesicular space was temperature dependent and independent of sodium. TC uptake (2 microM) was markedly inhibited by 250 microM taurine and glycine-conjugated cholate and chenodeoxycholate and unconjugated cholate but not by chenodeoxycholate, deoxycholate, etianic acid, bromosulfophthalein, pyruvate, lactate, alanine, or taurine. The initial rate of TC uptake was inhibited significantly by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) but was not inhibited significantly by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, amiloride, or furosemide. Preincubation of vesicles with DIDS in the presence of TC partially blocked the action of the inhibitor. Efflux of 5 microM TC from membrane vesicles was stimulated by the presence of 50 microM TC in the incubation media. Basal as well as transstimulated TC efflux was inhibited by DIDS. The initial rate of TC influx followed saturation kinetics with an apparent Michaelis constant of 112 +/- 23 microM and maximal velocity of 2.01 +/- 0.19 nmol.mg protein-1.min-1. When the transmembrane electrical potential difference across the brush-border membrane vesicles was altered by external anion replacement or by valinomycin-induced K+ diffusion potentials, TC uptake was not significantly affected. DIDS-sensitive TC uptake was stimulated two-to threefold by an outwardly directed hydroxyl gradient (pH 7.7in/5.5out) compared with TC influx under pH-equilibrated conditions (pH 7.7in/7.7out). These studies are consistent with an electroneutral anion-exchange mechanism that mediates transfer of conjugated bile acids across the microvillous membrane of the syncytiotrophoblast.

Reversal of glibenclamide and voltage block of an epithelial KATP channel

1996 ◽  
Vol 271 (4) ◽  
pp. C1122-C1130 ◽  
Author(s):  
O. Mayorga-Wark ◽  
W. P. Dubinsky ◽  
S. G. Schultz
Keyword(s):  
Hydrophobic Interactions ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Voltage Gating ◽  
Electrical Potential Difference ◽  
Outer Solution ◽  
Channel Inhibition ◽  
K Concentration ◽  
Voltage Gate

K+ channels present in basolateral membrane vesicles isolated from Necturus maculosa small intestinal cells and reconstituted into planar phospholipid bilayers are inhibited by MgATP and sulfonylurea derivatives, such as tolbutamide and glibenclamide, when these agents are added to the solution bathing the inner mouth of the channel. In addition, these channels possess an intrinsic "voltage gate" and are blocked when the electrical potential difference across the channel is oriented so that the inner solution is electrically positive with respect to the outer solution. We now show that increasing the concentration of permeant ions such as K+ or Rb+ in the outer solution reverses channel inhibition resulting from the addition of 50 microM glibenclamide to the inner solution and also inhibits intrinsic voltage gating; these effects are not elicited by increasing the concentrations of the relatively impermeant ions, Na+ or choline, in the outer solution. Furthermore, increasing the K+ concentration in the outer solution in the absence of glibenclamide inhibits voltage gating, and, under these conditions, the subsequent addition of glibenclamide to the inner solution is ineffective. These results are consistent with a model in which the voltage gate is an open-channel blocker whose action is directly reversed by elevating the external concentration of relatively permeant cations and where the action of glibenclamide is to stabilize the inactivated state of the channel, possibly through hydrophobic interactions.

Phosphate transport in kidneys: effect of transmembrane electrical potential

1991 ◽  
Vol 261 (4) ◽  
pp. F663-F669 ◽  
Author(s):  
R. Beliveau ◽  
J. Strevey
Keyword(s):  
Driving Force ◽  
Transmembrane Potential ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Saturation Curve ◽  
Maximal Velocity ◽  
Transmembrane Electrical Potential ◽  
The Hill ◽  
Phosphate Influx

The effect of a transmembrane electrical potential on phosphate transport by kidney brush-border membrane vesicles was studied. The initial rate of Na(+)-dependent phosphate influx was twice as high as that of efflux. Generation of a negative transmembrane potential had a stimulatory effect on the rate of influx but had no effect on efflux. The Na+ saturation curve for phosphate influx was sigmoidal, and the Hill coefficients were similar, in the presence and absence of a transmembrane potential. The membrane potential increased both the affinity for phosphate and the maximal velocity (Vmax) of the transporter. In the absence of a Na+ gradient, the stimulation by the potential was 1.78-fold. When a proton gradient (in greater than out) was the driving force, the electrical potential stimulated phosphate transport 1.71-fold. Internal Na+ (trans) inhibited phosphate influx whether a potential was present or not. Internal phosphate (trans) stimulated phosphate influx in the absence of a potential but not in its presence. These results indicate that the electrical potential is an important driving force for the Na(+)-phosphate carrier and that the translocation of the carrier is a potential-dependent step.

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