Secondary Transport Systems Research Articles

Interpretation of steady-state current-voltage curves: Consequences and implications of current subtraction in transport studies

A problem often confronted in analyses of charge-carrying transport processes in vivo lies in identifying porter-specific component currents and their dependence on membrane potential. Frequently, current-voltage (I-V)--or more precisely, difference-current-voltage (dI-V)--relations, both for primary and for secondary transport processes, have been extracted from the overall membrane current-voltage profiles by subtracting currents measured before and after experimental manipulations expected to alter the porter characteristics only. This paper examines the consequences of current subtraction within the context of a generalized kinetic carrier model for Class I transport mechanisms (U.-P. Hansen, D. Gradmann, D. Sanders and C.L. Slayman, 1981, J. Membrane Biol. 63:165-190). Attention is focused primarily on dI-V profiles associated with ion-driven secondary transport for which external solute concentrations usually serve as the experimental variable, but precisely analogous results and the same conclusions are indicated in relation to studies of primary electrogenesis. The model comprises a single transport loop linking n (3 or more) discrete states of a carrier 'molecule.' State transitions include one membrane charge-transport step and one solute-binding step. Fundamental properties of dI-V relations are derived analytically for all n-state formulations by analogy to common experimental designs. Additional features are revealed through analysis of a "reduced" 2-state empirical form, and numerical examples, computed using this and a "minimum" 4-state formulation, illustrate dI-V curves under principle limiting conditions. Class I models generate a wide range of dI-V profiles which can accommodate essentially all of the data now extant for primary and secondary transport systems, including difference current relations showing regions of negative slope conductance. The particular features exhibited by the curves depend on the relative magnitudes and orderings of reaction rate constants within the transport loop. Two distinct classes of dI-V curves result which reflect the relative rates of membrane charge transit and carrier recycling steps. Also evident in difference current relations are contributions from 'hidden' carrier states not directly associated with charge translocation in circumstances which can give rise to observations of counterflow or exchange diffusion. Conductance-voltage relations provide a semi-quantitative means to obtaining pairs of empirical rate parameters.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional incorporation of beef-heart cytochrome c oxidase into membranes of Streptococcus cremoris.

Beef heart mitochondrial cytochrome c oxidase has been incorporated into membrane vesicles derived from the homofermentative lactic acid bacterium Streptococcus cremoris. Proteoliposomes containing cytochrome c oxidase were fused with the bacterial membrane vesicles by means of a freeze/thaw sonication technique. Evidence that membrane fusion has taken place is presented by the demonstration that nonexchangeable fluorescent phospholipid probes, originally present only in the bacterial membrane or only in the liposomal membrane, are diluted in the membrane after fusion and, by sucrose gradient centrifugation, indicating a buoyant density of the membranes after fusion in between those of the starting membrane preparations. The fused membranes are endowed with a relatively low ion permeability which makes it possible to generate a high proton motive force (100 mV, inside negative and alkaline) by cytochrome-c-oxidase-mediated oxidation of the electron donor system ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c. In the fused membranes this proton motive force can drive the uptake of several amino acids via secondary transport systems. The incorporation procedure described for primary proton pumps in biological membranes opens attractive possibilities for studies of proton-motive-force-dependent processes in isolated membrane vesicles from bacterial or eukaryotic origin which lack a suitable proton-motive-force-generating system.

1] Energetics of proton transport and secondary transport

Publisher Summary This chapter discusses energetics of proton transport and secondary transport. Proton pumps and their associated proton-coupled transport systems are the most common biological systems of energy conversion. In describing the energetics of these systems, it is useful to distinguish between primary active transport systems or pumps, in which transport of an ion is directly coupled to a chemical (or photochemical) reaction, and secondary active transport systems, in which the vectorial transport of ions is coupled to the transport of other ions and substrates. The existence of several primary and secondary active transport systems which share common ions and substrates in the same membrane leads to complex energy conversion pathways. This chapter discusses the energetics of individual transport systems. However, as it is not always possible to study the energetics of a totally isolated system (except perhaps in a reconstituted system) the existence and the activity of other systems in the same membrane must be accounted for in a quantitative description of the energetics of primary and secondary transport.

H +/ion antiport as the principal mechanism of transport systems in the vacuolar membrane of the yeast Saccharomyces carlsbergensis

The secondary transport systems of the yeast vacuolar membrane have been investigated by (i) the method of radioactive isotopes ([ 14C]arginine); (ii) activation of H +-ATPase by cations (Cat +), when the enzyme is under H + control and (iii) measurement of changes in the proton gradient (Δ pH) and membrane potential ( E m) due to the supposed substrates of the transporters. The main mechanism of cation transport across the yeast tonoplast is probably H +/Cat + antiport. The apparent K m of antiporters for Ca 2+, Mg 2+ Mn 2+, Zn 2+ and P i are 0.06, 0.3, 0.8, 0.055-0.17 and 1.5mM, respectively.

Calcium transport in membrane vesicles ofStreptococcus cremoris

Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A. J. M.. Hellingwerf, K. J. & Konings, W. N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing AI~ with a concomitant increase in ApH stimulates Ca2+ uptake while dissipation of the ApH results in a reduced rate of Ca2+ uptake. Also an artificially generated ApH, interior acid, can drive CaZ+ uptake in S. cremoris membrane vesicles. CaZ+ uptake depends strongly on the presence of external phosphate while Ca'+-efflux-induced proton flux is independent of the presence of external phosphate. Caz+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,W-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons.

H+-Translocating ATPases: Advances Using Membrane Vesicles

In this paper, two primary active transport systems (H/sup +/ -ATPases) in plant cells are examined using membrane vesicles as a simple experimental tool. One electrogenic, H/sup +/ -translocating ATPase is vanadate-sensitive and associated with the plasma membrane. Another electrogenic, H/sup +/ -translocating ATPases is anion-sensitive, and localized on the tonoplast (and perhaps other membranes). According to the working model, the plasma membrane and tonoplast-type H/sup +/ -ATPases are detectable in inside-out plasma membrane and right-side-out tonoplast vesicles. The direction of H/sup +/ pumping into these vesicles would be consistent with the results from intact cells where H/sup +/ are extruded from the cell across the plasma membrane and pumped into the vacuole from the cytoplasm. Understanding the properties of H/sup +/ -pumping ATPases using membrane vesicles has paved the way for studies to identify secondary active transport systems coupled to the proton electrochemical gradient. Redox-driven transport systems can also be studied directly using the isolated vesicles. As transport proteins are identified, the functional activities can be specifically studied after reconstitution of the purified protein(s) into phospholipid membrane vesicles. 154 references.

Sodium-dependent transport of inorganic sulfate by rabbit renal brush-border membrane vesicles. Effects of other ions.

A Na+ gradient (extravesicular greater than intravesicular) increased the rate of inorganic sulfate (SO24-) uptake into renal brush-border membrane vesicles and energized the transient accumulation of the anion against its concentration gradient, indicating a secondary active transport system. Stimulation of SO24- uptake was specific for Na+. The anions, SO23-, S2O23-, SeO24-, MoO24-, CrO24-, and WO24-, but not HPO24-, cis-inhibited and trans-stimulated SO24- uptake, suggesting that these divalent anions shared the SO24- carrier. The Na+/SO24- co-transport and Na+. The apparent Km for SO24- was 0.6 mM at 100 mM Na+. The relationship between Na+ concentration and rate of SO24- uptake was sigmoidal. From a Hill analysis of the data a [Na+]0.5 of 36 mM and an n value of 1.6 were calculated. Comparisons of the effects of a K+ diffusion potential (inside positive), of a H+ diffusion potential (inside negative), of Na+ salts of anions of different conductances on the Na+-dependent uptakes of SO24- and D-glucose, and of the responses of a membrane potential-sensitive fluorescent probe concomitant with the uptakes indicate that Na+/SO24- co-transport was electroneutral. The simplest stoichiometry consistent with an electroneutral mechanism would be the co-transport of two Na+ and one SO24-. Na+ gradient-dependent SO24- uptake was enhanced by intravesicular Cl-. cis-Cl- inhibited the efflux as well as the influx of SO24-. These findings suggest that Cl- was an inhibitor of SO24- transport. Intravesicular K+ stimulated Na+ gradient-dependent SO24- uptake. The co-transport of Na+/SO24- appeared not to be coupled to the transmembrane flux of K+. It is hypothesized that the co-transport system contained an internal site activated by K+.

Electrogenic systems of a cell and membrane permeability

The equation of membrane potential (Δψ) for electrogenic cation pump operation has been developed. The expression obtained can be equally used for the description of Δψ created by any electrogenerator that transduces light or chemical energy into electrochemical potential difference. The equation reflects the asymmetric manner of charge transfer across such systems. On the basis of the equation and of the equivalent electrical circuit the simple qualitative analysis of interplay between electrogenic and electro-utilizing systems is considered. The conclusion is that the Δψ cannot exceed the definite value for the following reasons: 1. The metabolic energy which is employed for Δψ generation is not more than 40–50 kJ. In accordance with the first law of thermodynamics the Δψ will also have a limiting value since a further increase of Δψ would lead to self-blocking of the electrogenerator. 2. The occurrence of secondary transport and diffusion systems which utilize the energy of the electrical field permanently decrease Δψ. 3. The electrical field causes microruptures inside the membrane and makes for the formation of novel leakage channels.

Electrogenic systems of a cell and membrane permeability

Summary The equation of membrane potential (Δψ) for electrogenic cation pump operation has been developed. The expression obtained can be equally used for the description of Δψ created by any electrogenerator that transduces light or chemical energy into electrochemical potential difference. The equation reflects the asymmetric manner of charge transfer across such systems. On the basis of the equation and of the equivalent electrical circuit the simple qualitative analysis of interplay between electrogenic and electro-utilizing systems is considered. The conclusion is that the Δψ cannot exceed the definite value for the following reasons: o (1) The metabolic energy which is employed for Δψ generation is not more than 40–50 kJ. In accordance with the first law of thermodynamics the Δψ will also have a limiting value since a further increase of Δψ would lead to self-blocking of the electrogenerator. (2) The occurrence of secondary transport and diffusion systems which utilize the energy of the electrical field permanently decrease Δψ. (3) The electrical field causes microruptures inside the membrane and makes for the formation of novel leakage channels.

Conversion of the chemical energy of methylmalonyl-CoA decarboxylation into a Na+ gradient.

Primary active transport systems establish electrochemical gradients of their transport substrates at the direct expense of chemical or light energy. The chemical energy can be provided by cleavage of the phosphoric anhydride bond of ATP, establishing Na+, K+, H+ or Ca2+ gradients with the respective ATPases. Whereas in eukaryotic cells the Na+ and K+ imbalance between cells and the surrounding fluids is brought about by the (Na+ + K+)ATPase1, no such enzyme is known for bacterial cells. Generally, the gradients of Na+ and K+ ions in these cells are believed to be generated by secondary transport systems consuming the energy provided by the electrochemical gradient of protons2,3. In some bacterial species, however, sodium ions may be transported by primary ATP or light-driven sodium pumps4,5. The primary sodium pump oxaloacetate decarboxylase represents a novel energy conversion mechanism in that it consumes the chemical energy of the decarboxylation of oxaloacetate to drive Na+ transport6–8. We show here that methylmalonyl-CoA decarboxylase is another example of a Na+ transport enzyme converting the chemical energy of a decarboxylation reaction into an ion gradient.

Primary and secondary transport systems for amino acids in the intact intestine.

IN OUR PREVIOUS INVESTIGATIONS we have been able to demonstrate the net and absolute absorption rates for amino acids from the small intestine in the intact rat. 1 We also demonstrated that absorption could be inhibited by inducing pyridoxine deficiency and by the antimetabolites deoxypyridoxine and 2, 4-dinitrophenol (DNP). Alleviation of this inhibition was dependent upon the B6-vitamin factor in the form of pyridoxal-5-phosphate. 1,2 The problem with which we were confronted was to investigate whether or not this inhibition of mediated intestinal absorption was one in which the cells of the mucosa were affected so that they could not selectively take up the amino acid, or could accept a normal load but not pass this on to the circulation. These experiments are intended to show that the inhibited absorption is involved in what we might postulate as a secondary transport system from the loaded intestinal cells rather than