2.4.5—
Membrane Carriers

Many ions and uncharged solutes cross membranes more rapidly than could be expected if they passed through the membrane lipids without some assistance. Since pores in membranes are too narrow to accommodate many substances which are transported, it is widely believed that membranes contain carrier molecules which, in combination with the solute, facilitate diffusion. The ion pumps considered above are carriers of a special kind since they are linked to

the metabolic activity of the cell; the carriers we shall now consider promote net movements of solutes only down the prevailing potential gradient and are not capable of 'uphill' transport even if in some instances (see p. 57) they appear to be doing so.

2.4.5.1—
Evidence from Kinetics

One widely used approach to gather information about carriers has been the application of kinetics first derived from enzyme reactions. The evidence for these carriers is obtained by placing a cell or tissue in a range of solute concentrations and measuring the initial rate of uptake. As illustrated in Fig. 2. 15 the uptake rate shows a tendency to saturate at higher concentrations and can thus be used to calculate the maximum velocity, V_max , possible under the conditions used in the experiment. By making a double reciprocal plot of the data (Fig. 2.15) the concentration of solute at which half maximal velocity is achieved can

Figure 2.15
Saturation kinetics of solute uptake versus concentration. Such results are used
as evidence for the association of the solute and a carrier. The double reciprocal plot of
the data gives a more accurate estimate of  V_max  and K_m  when the number of points is limited.

be estimated. This is known as the Michaelis Constant, K_m . The K_m measures the affinity of the carrier for the solute it carries; if the affinity is high then the concentration, K_m will be low and vice versa. For most ions in plant tissue K_m is quite low, concentrations ranging from 5–100 mM , but for sugars and other metabolites K_m values are usually greater than 300 mM . Much work of this kind is summarized by Epstein (1972) who shows that at concentrations less than 0.1 mM , the uptake of a given ion is not subject to serious interference from other

common ions in solution. There is, however, competitive inhibition between related ions of similar molecular dimensions, e.g. K⁺ uptake is inhibited competitively by Rb⁺ but not by Na⁺ ; Ca²⁺ is inhibited by Sr²⁺ but not by Mg²⁺ . Thus the carriers which bind the major nutrient ions at low concentrations appear to be highly ion-selective. At higher concentrations (more than 1.0–10.0 mM ) this selectivity begins to decline. The interpretation of this observation is contentious and beyond the scope of this chapter but can be pursued in Epstein (1972), Laties (1969) and Clarkson (1974).

The limitation of the kinetic approach is that it can tell us nothing about the nature of the carrier. One can observe similar uptake kinetics for ions whose transport into the cell must be mediated by ion pumps e.g. H₂ PO₄ ^– and Cl^– (see p. 48) as for ions which probably diffuse into the cell passively e.g. Na⁺ and Ca²⁺ and for those which are completely exotic and toxic, e.g. Tl⁴⁺ (Barber, 1974). Indeed, it has been pointed out that saturation kinetics of this kind would also be found if salt movement was observed across a synthetic membrane containing nothing but pores (Stein & Danielli, 1956), where the system would saturate when all of the pores were filled with solute at any moment in time; V_max is, after all, merely a measurement of capacity to react or transport and K_m is derived from it (Fig. 2.15).

2.4.5.2—
Ionophores as Lipophilic Carriers

A more illuminating approach to the nature of carriers has come from studies on the ionic conductance of synthetic membranes which have been modified in various ways. A bilayer of pure phospholipids has a very low conductance to ions, usually only 10^–7 to 10^–8 ohm^–1 cm^–2 . The addition of very small amounts of ionophores (i.e. ion-carrying antibiotics) like monactin or valinomycin to the solutions bathing the synthetic bilayer causes a huge increase in the conductance. Figure 2.16 shows that 10^–6M monactin changes the membrane conductance to K⁺ nearly a million-fold and that even at 10^–10 M its effect is quite strong. The conductance change for a given monactin concentration is greatest for K⁺ and for Rb⁺ and is much less for Na⁺ , Cs⁺ and Li⁺ . Monactin is, therefore, acting as a selective carrier of K⁺ and Rb⁺ and valinomycin, another bacterial product, behaves similarly. Since these two compounds differ chemically it is instructive to see what they have in common. Both of them are amphipathic ring-structured molecules which have their non-polar groups on the outside of the ring and their polar groups directed towards the space at the centre of the molecule. The outside of the ring interacts favourably with lipid while the hydrophilic core, 0.7 nm in diameter, provides room for several hydrated potassium ions to be bound. Evidence from a variety of sources shows that this complex diffuses across the membrane so that the ions never leave a polar environment (Eisenman et al., 1968).

Other substances are known which select for divalent cations, e.g. the unnamed compound A23187 which is a carboxylic acid antibiotic found in

Figure 2.16
Influence of the ionophore, monactin, on the electrolytic conductance of a
phospholipid bilayer in the presence of single salt solutions of alkali cations.
(Redrawn from Eiseman et al.,  1968.)

cultures of Streptomyces chartreusensis (Reed & Lardy, 1972). This compound carries Ca²⁺ and Mg²⁺ across bilayers and natural membranes but has no effect on monovalent cations.

Apart from a few synthetic analogues all of the ion-carrying antibiotics are natural products of bacteria and fungi. There are many who believe that compounds of a similar kind may act as ion carriers in all membranes but the technical difficulty of isolating what are probably minute quantities of such compounds from tissues appears to be formidable and so the belief may rest on faith for some time yet.

2.4.5.3—
Co-transport

As suggested earlier, carrier-assisted diffusion can sometimes appear to go in an 'uphill' direction, thus giving the impression of active transport. In many animal tissues and micro-organisms, sugars, amino acids, organic acids and vitamins move into the cell up a concentration gradient. This transport is, however, almost completely dependent on having Na⁺ or H⁺ in the external medium; other ions such as K⁺ , Rb⁺ or Li⁺ cannot be substituted. It has been

found that the metabolite is carried into the cell along with an ion-carrier complex which is diffusing 'downhill' (Fig. 2.17). In both Chlorella and Neurospora, glucose is transported in this way along with protons, H⁺ (Komor & Tanner, 1974; Slayman & Slayman, 1974).

Figure 2.17
Scheme to illustrate co-transport of protons and sugar. The proton extrusion pump is electrogenic
and thus makes the inside electrically negative. Protons diffuse back into the cell passively  via  the
carrier which also binds a sugar molecule. The protonated carrier plus sugar diffuses towards the
inner face of the membrane where it dissociates and releases the sugar molecule.

Co-transport depends on active transport in an indirect way (as indeed does all diffusion, see p. 50) because energy-dependent extrusion pumps ensure that the cytoplasm is kept well below its equilibrium concentration in H⁺ and Na⁺ . These ions tend to diffuse back into the cell and, in doing so, decrease their free energy. The energy they give up is coupled, via the carrier, to the co-transport of the solute whose free energy is increased as it moves into the cell.

Co-transport may also assist the 'uphill' movement of inorganic ions into the cell; it may be a more common process than is generally realized. Recent

evidence by Lowendorf et al., (1974) suggests that the active transport of phosphate into the hyphae of Neurospora depends on (a) the activity of a proton extrusion pump at the plasmalemma which is sensitive to the pH of the external medium, and (b) the formation of a ternary complex between a proton and a phosphate ion from the external medium with a membrane carrier. The protonated phosphate carrier diffuses to the cytoplasmic side of the membrane down the electrochemical gradient of the proton, releasing the proton and phosphate ion into the cytoplasm. This, and other examples of inorganic ion co-transport (see Raven & Smith, 1974) suggest a reason why so little progress has been made in elucidating the molecular details of certain 'pumps' particularly of those which transport anions. Put most simply, it may be that these influx pumps do not exist and that the uphill transport is driven by a combination of active extrusion and re-entry by diffusion of protons or perhaps sodium ions.

Co-transport illustrates the ingenious way in which nature can turn necessity to its own advantage. The active excretion of H⁺ and Na⁺ is essential to maintain pH control and osmo-regulation in the cell, but the energy expended is partly recovered in the transport of essential metabolites and ions into the cell.