Ian C. West

Sodium-lithium countertransport : physiology and function

Current opinions on the relationships between erythrocyte sodium-lithium countertransport kinetics and primary hypertension, hyperlipidaemia and diabetic nephropathy are reviewed. Problems associated with the assay are analysed. Some possible mechanisms that could modify the kinetics of ion exchange are examined. The question of what catalyses sodium-lithium countertransport is discussed, but not answered. Some models are put forward showing how a study of sodium-lithium countertransport kinetics could further our understanding of important disease processes.

What determines the substrate specificity of the multi-drug-resistance pump?

Multi-drug-resistance protein (P-glycoprotein) turns out to be an ATP-hydrolysing transmembrane pump that increases the resistance of cells in which it is expressed by actively extruding toxic chemicals. The baffling question is how does the pump know which chemicals to extrude? Common features among its substrates are still elusive. The question raised here concerns the relationship between this pump and that known for many years as capable of extruding glutathionyl and cysteinyl S-conjugates of xenobiotics. Are excreted drugs conjugated before excretion? Does the multi-drug-resistance pump recognize a simple chemical tag put on xenobiotics by a family of transferase enzymes?

Sulphydryl group control of sodium-lithium countertransport kinetics: a membrane protein control abnormality in essential hypertension

Abstract. Erythrocyte sodium‐lithium countertransport (SLC) is an obligatorily coupled equimolar exchange of intracellular sodium or lithium with extracellular sodium or lithium. SLC is partially inhibited by N‐ethylmaleimide (NEM) but only when a transported ion (sodium or lithium) is present in the extracellular medium. In essential hypertensive patients with a strong family history of hypertension the Km of SLC for extracellular sodium was lower and Vmax tended to be higher than in normal controls, but the ratio Vmax/ Km gave a much clearer distinction between the two groups. After NEM treatment, the remaining SLC activity in normal individuals had a lower Vmax and Km for sodium but Vmax/Km was not affected. In essential hypertensives the remaining SLC activity after NEM again had lowered Vmax and Km but in these patients the Vmax/Km was much lower than in untreated erythrocytes and was then the same as in normal controls. On the assumption that NEM reacts with a ‐SH group on a membrane protein that regulates SLC, and that the ratio Vmax/Km reflects a rate constant for binding extracellular sodium to the unloaded carrier, the results suggest that (a) essential hypertensives have an increased rate of sodium binding to the transporter and (b) this is due to abnormal behaviour of a membrane ‐SH group.

When is the outer membrane of Escherichia coli rate-limiting for uptake of galactosides?

During inflow into Escherichia coli substrates must first diffuse through the porin pores in the outer membrane by simple, passive, diffusion and then be translocated across the inner membrane by a specific (active) carrier protein, or permease. A graphical procedure is outlined whereby it is possible to estimate the concentration drop across the outer membrane from simple kinetic measurements of net inflow velocity. Experiments confirm that the concentration drop across the outer membrane is proportional to rate of inflow, as expected from Ficks law. The expected rate of diffusion of 2-nitrophenylgalactoside through the outer membrane was calculated from reported values of pore radius, length and number, and the rate was found to correspond closely with the experimental results. It is pointed out that at low substrate concentrations the outer membrane is rate-limiting and that a large increase in the amount of permease in the inner membrane will cause very little change in net inflow velocity.

Modification of erythrocyte Na+/Li+ countertransport kinetics by two types of thiol group

Erythrocyte Na+/Li+ countertransport activity is decreased by reagents that react with thiol groups. An understanding of the role of these groups in control of Na+/Li+ countertransport may help to explain its association with disease states. The effect of thiol reactive agents on the kinetic parameters of Na+/Li+ countertransport has not previously been described. In choline medium, N-ethylmaleimide (NEM) and iodoacetamide (IAamide) cause a rapid decrease of about 40% in Km for external sodium (Km(So)) that is complete in 10 s with a much smaller change in Vmax and an increase in the Vmax/Km ratio. In Na medium, NEM and IAamide both cause a rapid decrease in Km(So) and Vmax. With NEM the partial reduction in Vmax is complete in 100s although the NEM is sufficient to reduce Vmax up to 15 min. With IAamide the decrease in Vmax is initially slower but it continues apparently towards complete inhibition. These results indicate at least two types of thiol group controlling Na+/Li+ countertransport kinetics. The type 1 thiol reaction is Na independent and causes an increase in the apparent rate constant for Na association with the unloaded carrier so that Vmax/Km rises and Km(So) decreases. The type 2 thiol reaction is facilitated by Na at the outside ion-binding site and causes a decrease in Vmax, possibly by total blockage of carriers with IAamide but by a different mechanism with NEM such as reduced turnover rate.

Abnormal thiol reactivity of tropomyosin in essential hypertension and its association with abnormal sodium-lithium countertransport kinetics

Objectives To identify a thiol protein that is abnormal in a subgroup of essential hypertensive (EHT) patients who have a strong family history of hypertension and cardiovascular disease and have a low Km of erythrocyte Na/Li countertransport (CT). Methods To detect biotin maleimide labelling of a key thiol protein to investigate its reaction with N-ethylmaleimide (NEM) in normal and EHT erythrocytes. Results The thiol protein of 33 kDa apparent molecular weight (p33) identified by the loss of labelling with biotin maleimide was identified as tropomyosin due to its retarded running in 6 mol/l urea gels and immunoblotting. The NEM reaction with p33 detected by loss of subsequent biotin maleimide labelling is biphasic in normal control erythrocytes with the rate in the first 30 s double that after 30 s. In EHT erythrocytes NEM reaction (1) after 30 s is faster than normal and (2) in the first 30 s causes a paradoxical increase in apparent biotin maleimide labelling. In normal control erythrocytes, the loss of biotin maleimide labelling with NEM reaction or the faster phenylmaleimide reaction follows the same time course as the decrease in Km of Na/Li CT. Conclusions NEM reaction with p33 requires two thiols. Only the cytoskeletal form of tropomyosin from the TM3 gene has more than one thiol group and agrees with SDS-PAGE mobility. Tropomyosin is a strong candidate to explain the familial abnormality in EHT with abnormal Na/Li CT and it could explain many of the characteristics of this disease.

Kinetics of lactose transport into Escherichia coli in the presence and absence of a protonmotive force

The kinetics of galactoside movements into and out of whole cells of Escherichia coli under metabolizing and metabolically-inhibited conditions were described by Winkler and Wilson [ 11. They found that the inhibition of metabolism caused the very high app. Km for efflux to fall to the lower values characteristic of the app. Km for infhrx, whereas the app. Km for influx was not detectably changed. These results were substantially confirmed in [2], but in a neglected paper [3], the presence of uncoupler was reported to cause the app. Km for influx of thiomethylgalactoside to increase to a much higher value. In the presence of a protonmotive force membrane vesicles accumulate lactose with an app. Km similar to that for influx into whole cells [5,6]. In the absence of added respirable substrate there is essentially no protonmotive force and galactosides are not accumulated, though they may still bind to the carrier [6]. Under the latter conditions, the binding constant for lactose was 200-fold greater than the app. Km for active uptake [6]. Similar results have been reported in [7]. This paper describes experiments designed to examine the kinetics of the lactose carrier in the absence of a protonmotive force but under conditions of net transport. The results rule out the possibility that the difference between the app. Km and Kd observed in [6] could be due to measurements being made under different conditions (i.e., that the app.

Characterisation in vivo of the reactive thiol groups of the lactose permease from Escherichia coli and a mutant; exposure, reactivity and the effects of substrate binding

The reactivity and accessibility of the reactive thiol groups of the native lactose permease and a mutant have been studied in a number of circumstances and with a number of reagents, in particular using the specific thiol-disulphide exchange reaction. Seven different reactive states of the thiol in the native protein have been characterised by their different second-order rate constants. Interconversion between these states is dependent on the magnitude of the protonmotive force, pH and substrate binding. In the absence of galactoside, reactivity is controlled by an ionisation with apparent pKa 9.3. This pKa is not affected by the protonmotive force, but it is lowered in the presence of external galactoside. The conformation adopted by the permease when in equilibrium with saturating galactoside appears to be different from that of the intermediate that accumulates during net turnover. In the former state, the reactivity of the thiol group is depressed, whereas in the latter state it is enhanced. The thiol group of the native protein is buried in a hydrophobic environment that has a dielectric constant considerably lower than that of water. The environment is not greatly perturbed by changes in the magnitude of the protonmotive force, but it is affected by the binding of galactoside. In a strain which carries the YUN mutation (Wilson, T.H. and Kusch, M. (1972) Biochim. Biophys. Acta 255, 786-797), two reactive thiols were characterised. The more reactive of the two is more exposed than the thiol group of the native molecule and is in an environment that has a dielectric constant close to that of water. The less reactive thiol appears to be more deeply buried than that of the native protein. Thus the mutation appears to produce a conformation change in the central portion of the polypeptide chain that results in greater exposure of the reactive thiol to the aqueous environment.

Thiol Group Control of Sodium-Lithium Countertransport Kinetics in Uraemia: Evidence of a Membrane Abnormality Affected by Haemodialysis

Uraemia affects erythrocyte metabolism and membrane function but no consistent effect on Na/Li countertransport (CT) has been reported. We report only small differences in Na/Li CT at 150 mmol/l Na over haemodialysis, but major differences in other properties of Na/Li CT. The Km for external sodium and Vmax both increased during haemodialysis but the Vmax/Km ratio, which was greater than normal, was not affected. The thiol reagent, N-ethylmaleimide (NEM), which causes a decrease in Km and Vmax in normal subjects, had no effect on Km in the predialysis erythrocytes. After haemodialysis, the sensitivity of Na/Li CT to NEM was improved. The changes in Na/Li CT kinetics were not related to changes in membrane lipid fluidity or plasma lipids. These observations suggest that uraemia affects a thiol group that controls Na/Li CT kinetics and that haemodialysis temporarily improves this aspect of membrane function.

General principles of membrane transport

Publisher Summary This chapter discusses the general principles of membrane transport. Active transport—the transport of solutes (either inward or outward) from a lower to a higher electrochemical potential—can be achieved, and can only be achieved, by coupling the free-energy consuming transport process to a free-energy yielding process. This may be a chemical reaction for which the free-energy change is negative—for example, the hydrolysis of adenosine triphosphate (ATP) by water to adenosine diphosphate (ADP) and inorganic phosphate (P i ), in which case the combined (coupled) process is described as primary active transport. The free-energy yielding process may also be another transport reaction—for example, the flow of Na + (or H + ) down its electrochemical potential gradient into the animal (or plant, or bacterial) cell. It is quite helpful to emphasize (to oneself) that the two parts of the combined (coupled) process do not actually exist in themselves as separate entities with the energy mysteriously travelling from one to the other. There are pores, or channels, in the plasma membranes of many cell types in which a considerable degree of selectivity is observed, and where the pore is by no means always open. However, though these regulated channels may show open and closed states, they nevertheless do not close and open between each transport event.