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.

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.

scholarly journals Electrogenic 2Na+/H+ Antiport in Echinoderm Gastrointestinal Epithelium

1991 ◽  
Vol 158 (1) ◽  
pp. 495-507 ◽  
Author(s):  
GREGORY A. AHEARN ◽  
PIERETTE FRANCO
Keyword(s):  
Membrane Potential ◽  
Driving Forces ◽  
Flux Ratio ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Proton Gradient ◽  
Hill Coefficient ◽  
Sodium Influx ◽  
22Na Uptake ◽  
Static Head

Purified brush-border membrane vesicles (BBMV) of starfish (Pycnopodia helianthoides) pyloric caecal epithelium were prepared by a magnesium precipitation technique in order to compare the properties of Na+/H+ exchange in this invertebrate tissue with those of an apparently unique recently described crustacean electrogenic antiporter. In starfish BBMV 22Na uptake was markedly enhanced by an outwardly directed pH gradient and membrane potential (inside negative) compared to control short-circuited vesicles. External amiloride abolished the stimulatory capacity of the proton gradient and membrane potential as driving forces for sodium transport. Sodium influx, in the presence of an outwardly directed proton gradient, was a sigmoidal function of [Na+]o and yielded a Hill coefficient of 2.6, suggesting that more than one sodium ion was exchanged with each internal proton during the exchange event. Two additional findings were used to establish the number of external Na+ binding sites and the transport stoichiometry of the starfish antiporter. First, amiloride acted as a competitive inhibitor of Na+ binding to two external sites with markedly dissimilar apparent amiloride affinities (Kil=28 μmoll−1; Kil=1650 μmoll−1). Second, a static head flux ratio analysis resulted in a 2Na+/H+ exchange stoichiometry where a balance of driving forces (e.g. no net Na+ flux) was attained with a combination of a 10:1 Na+ gradient and a 100:1 H+ gradient. Results suggest that the electrogenic 2Na+/H+ exchanger previously characterized for crustacean epithelia also occurs in echinoderm cells and may be a widely distributed invertebrate antiporter.

Electroneutral Na+/H+exchange in brush-border membrane vesicles fromPenaeus japonicus hepatopancreas

1998 ◽  
Vol 274 (2) ◽  
pp. R486-R493 ◽  
Author(s):  
Sebastiano Vilella ◽  
Vincenzo Zonno ◽  
Laura Ingrosso ◽  
Tiziano Verri ◽  
Carlo Storelli
Keyword(s):  
Kinetic Parameters ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Hill Coefficient ◽  
Uptake Kinetic ◽  
Ph Dependent ◽  
Transmembrane Electrical Potential

An electroneutral Na+/H+exchange mechanism (dimethylamiloride inhibitable, Li+ sensitive, and Ca2+ insensitive) was identified in brush-border membrane vesicles (BBMV) from Kuruma prawn hepatopancreas by monitoring Na+-dependent H+ fluxes with the pH-sensitive dye acridine orange and measuring22Na+uptake. Kinetic parameters measured under short-circuited conditions were the Na+ concentration that yielded one-half of the maximal dissipation rate ( F max) of the preset transmembrane ΔpH ( K Na) = 15 ± 2 mM and F max = 3,626 ± 197 Δ F ⋅ min−1 ⋅ mg protein−1, with a Hill coefficient for Na+ of ∼1. In addition, the inhibitory constant for dimethylamiloride was found to be ∼1 μM. The electroneutral nature of the antiporter was assessed in that an inside-negative transmembrane electrical potential neither affected kinetic parameters nor stimulated pH-dependent (intracellular pH > extracellular pH)22Na+uptake. In contrast, electrogenic pH-dependent22Na+uptake was observed in lobster hepatopancreatic BBMV. Substitution of chloride with gluconate resulted in increasing K Na and decreasing Δ F max, which suggests a possible role of chloride in the operational mechanism of the antiporter. These results indicate that a Na+/H+exchanger, resembling the electroneutral Na+/H+antiporter model, is present in hepatopancreatic BBMV from the Kuruma prawn Penaeus japonicus.

Kinetic analysis of electrogenic 2 Na+-1 H+ antiport in crustacean hepatopancreas

1989 ◽  
Vol 257 (3) ◽  
pp. R484-R493 ◽  
Author(s):  
G. A. Ahearn ◽  
L. P. Clay
Keyword(s):  
Binding Sites ◽  
Electrical Potential ◽  
Macrobrachium Rosenbergii ◽  
Cation Binding ◽  
Freshwater Prawn ◽  
Hill Coefficient ◽  
Affinity Constant ◽  
Electrical Potential Difference ◽  
Cation Site ◽  
S Uptake

Na+ uptake by short-circuited brush-border membrane vesicles of the hepatopancreatic epithelium from the freshwater prawn Macrobrachium rosenbergii was Cl- independent, amiloride sensitive, and stimulated by a transmembrane proton gradient ([H+]i greater than [H+]o). Na+ influx (3-s uptake) was a sigmoidal function of [Na]o (2.5-150 mM), when pHi = 6.0, pHo = 8.0, and followed the Hill equation for binding cooperativity [maximal Na+ influx (Jm) = 140.6 nmol mg-1s-1; affinity constant (K') = 82.2 mM Na+; Hill coefficient (n) = 2.07]. Influx kinetic analyses at physiological conditions suggested two external cation-binding sites shared by Na+ and H+ (proton dissociation constant Pk1 = 5.7; Pk2 = 4.0) and a single internal cation site used only by H+ (Pk = 6.5). Amiloride was a competitive inhibitor of Na+ transport at both external binding sites (Ki1 = 50 microM; Ki2 = 1,520 microM). Electrogenic Na+-H+ exchange by these vesicles was demonstrated using an equilibrium-shift method of analysis and a transmembrane electrical potential difference as the only driving force for transport. In addition, electrogenic net Na+ influx (3-s uptake) was observed in vesicles loaded with 5 mM 22Na at pH 7.0 and exposed to media containing several 22Na or proton concentrations. Results suggest the following exchange model: low [Na]o, (1 Na+ and 1 H+)-1 H+; high [Na+]o, 2 Na+-1 H+. This antiport mechanism may account for two major functional operations of the gastrointestinal tract in these animals: 1) proton secretion against considerable concentration gradients leading to stomach luminal acidification, and 2) Na+ absorption from lumen to cytoplasm potentially making a significant contribution to organismic ion balance.

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.

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.

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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