2.4.4—
Membrane Pumps

Pumping mechanisms which allow cells to accumulate solutes up gradients of potential can contribute to the membrane potential in both an indirect and a direct way. The former type are referred to as neutral exchange pumps in which the cell dumps an unwanted ion of equal and like charge into the external environment in a one-to-one exchange for an ion which is more useful, e.g. there are well known exchanges of cellular Na⁺ or H⁺ for K⁺ from the surroundings. The second type transports an ion in one direction only without coupled exchange and is known as electrogenic since charge is separated. They can, therefore add to, or subtract from the diffusion potential, described above, depending on which ion is carried. These two types of mechanisms are outlined in Fig. 2.11.

2.4.4.1—
Neutral Ion Pumps

Neutral ion pumps are present in the plasmalemma of all cells and by their activity they create the ionic asymmetry necessary to set up the diffusion potential described above. As a much simplified illustration of this, consider a cell which, because of its synthetic and respiratory activity, is generating H⁺ and HCO₃^– internally. A pair of exchange pumps could swap H⁺ and HCO₃^– for K⁺ and Cl^– from the surroundings quickly enriching the interior in these ions and thus setting up the conditions in which the differential rates of diffusion

of K⁺ and Cl^– out of the cell give rise to a membrane potential. But how does such a pump actually work? There are fewer detailed examples than one would like but the best known is of the membrane-bound ATPase which exchanges intracellular Na⁺ for extracellular K⁺ in many animal and plant cells (see Hall, 1971; Hodges et al., 1972). In the red blood cell it is known that Na⁺ is one of the cofactors which is essential for the binding of ATP to the ATP-ase enzyme. In vivo the active centre of the enzyme is accessible only from the cytoplasm, so that the ATP and the Na⁺ must be inside the cell (Fig. 2.14). Once bound, the ATP

Figure 2.14
A highly simplified illustration of the working of a sodium-potassium exchange pump based on a
membrane-bound ATPase. The cross hatched area on the ATPase is its active centre. The large
re-orientation of the molecule is for illustrative purposes only—quite subtle molecular re-arrangement
may be all that is necessary to expose the Na⁺ - binding site to the outside and for the step called relaxation.

is hydrolysed, ADP is released into the cytoplasm leaving the cleaved terminal phosphorus atom attached to the active centre to form a phosphoenzyme. These reactions result in some molecular re-orientation of the phosphoenzyme and its attached Na⁺ which exposes the ion-binding site to the different chemical environment of the external medium. It is proposed that this change of environment alters the ion-specificity of the binding site so that K⁺ is favoured; K⁺ thus replaces Na⁺ . This done, there is a second re-orientation (referred to as 'relaxation' in Fig. 2.14) which carries the bound K⁺ to the inside. The phosphorus is released from the active centre and Na⁺ , which is preferentially bound on the cytoplasmic side exchanges for K⁺ and the pump is ready for a second cycle. The pump has used the free energy released on hydrolysis of ATP as fuel to exchange K⁺ and Na⁺ against their respective electrochemical potential gradients. The two ions in the appropriate orientations are essential cofactors in the enzyme reaction; in vitro, ATPase of this kind will not hydrolyse ATP unless Na⁺ and K⁺ are both present.

In theory many pumps based on ATPase are possible with only subtle modifications of the ATPase molecule to provide binding sites of varying field

strength which will select various ions, e.g. a Ca²⁺ transporting ATPase is found in mitochondria and in sarcoplasmic reticulum (Racker, 1972).

2.4.4.2—
Electrogenic Pumps

The unidirectional transport of an ion across a membrane separates charges and in so doing provides a driving force for the passive diffusion of a similarly charged ion in the opposite direction or an oppositely charged ion in the same direction. The molecular details of exactly how an electrogenic pump is put together remain uncertain although in one instance it is highly likely that an electrogenic H⁺ -efflux pump is based on an ATPase (Slayman et al., 1973). It is possible, nevertheless, to deduce certain general consequences of their operation. If, for instance, there was an outwardly directed pump at the plasmalemma which actively pumped hydrogen ions (protons) out of the cell thus making the interior electrically negative, this could contribute to the electrical driving force on the diffusion of K⁺ from the external medium. Indeed the rate at which charge is extruded and the rate at which it leaks back into the cell must be very nearly in balance unless a dangerously large potential is to accumulate. Examples of both proton extrusion pumps and anion influx pumps of the electrogenic kind are well documented from research on plant tissues (Higinbotham & Anderson, 1974; Spanswick, 1972). In the giant alga, Acetabularia, an electrogenic chloride influx pump contributes more than half of the potential of –170mV found across the plasmalemma when the cell is kept in the light. Almost immediately the cell is put in the dark the pump stops working (since it is closely linked with photosynthesis) and the membrane potential abruptly depolarizes to –80mV (Saddler, 1970). A similar light-dependent electrogenic pump is found in Nitella translucens (Spanswick, 1972, 1974). Electrogenic pumps are not, however, restricted to green tissues but have been reported in plant roots (Higinbotham et al., 1970) and fungal hyphae (Slayman, 1970). In every instance, however, inhibition of the pump caused an immediate depolarization of the membrane potential, indeed this is often used to detect the activity of such a pump. The inhibition of a neutral ion pump gives rise to gradual depolarization as the ionic asymmetry runs down (see equation 2.6).