Some remarks on the integrated actions of pumps, cotransporters and channels

The biological significance of a particular molecular machine is defined only by its interactions with other molecular machines. While current research tends to emphasize direct interactions of macromolecules, indirect actions - often identified vaguely as ‘physiological’ functions or more specifically as transmitter- or messenger-mediated behavior - are clearly at least as important as direct interactions in the overall biological picture. What V-ATPases can accomplish becomes really interesting only when we know what they actually do accomplish; for example, in warehousing metabolic reserves, in compartmental buffering, in exporting adjuvant proteins and transmitter molecules and in processing nutrients. Wherever vacuolar-type ATPases appear, their action must be coordinated with actions of other transporters: i.e. with other pumps, co- or countertransporters, or bona fide channels; and with specific regulatory systems impinging on the same membranes.

Global variables which can modulate such interactions include membrane voltage, transmembrane chemical gradients, absolute solute composition (including pH) of cytoplasmic or extracellular fluids, redox potentials and, in some circumstances, luminous flux, temperature, etc. And much of transport biophysics has sprung from the fact that experimental values for such variables place essential thermodynamic constraints on the underlying reaction mechanisms.

To take a simple case in point, consider the question of how the free-living cells of many algae and fungi can concentrate nutrients from rarified environments to ratios of 10⁵–10⁶. One such nutrient is potassium, whose mode (or modes) of accumulative transport in non-animal cells, amazingly, still remains deeply in dispute; such cells clearly and categorically lack the P-ATPase-mediated exchange of K⁺ for Na⁺ which characterizes almost all animal cells (but which cannot sustain a concentration ratio much above 10⁴). Now, however, because patch electrode technology has opened up the examination of ion pores - or channels - as molecular entities, it has become fashionable to ascribe ‘almost all’ of K⁺ transport physiology in plants and fungi to the action of K⁺ channels. But chemical accumulation of ions via a passive pathway, such as a channel, requires coupling to either electrical or hydrodynamic flow; and the latter must be zero in a static, turgid plant or fungal cell (and small in any cell that survives long). The energetic equivalent of a concentration ratio of 10⁵–10⁶ is near 30 kJ mol⁻¹ or - for a univalent ion - about 300 mV. With a normal free energy of ATP hydrolysis less than 50 kJ mol⁻¹, any causally related ATP-driven charge pump could drive no more than one ion per ATP split (i.e. 1 mol of charge per mol of ATP). This constraint immediately rules out both V- and F-type ATPases as primary generators underlying putative channel-mediated K⁺ accumulation, since these enzymes function, as far as is known, with stoichiometries of 2 H⁺ (or perhaps more) per ATP split.

Animal physiologists and biochemists are often surprised to realize that plant and fungal cells can, and often do, sustain plasma membrane voltages approaching 300 mV. It is a necessary attribute of the free-living life style, and it has evolved with a P-type ATPase which indeed pumps a single ion (H⁺) for each ATP molecule hydrolyzed. The observations on accumulative potassium transport in plant and fungal cells, however, are even more severe. It is quite easy under laboratory conditions to produce stable accumulation ratios which exceed those allowed by measured membrane voltages acting through any kind of passive pathway. The inference drawn from this was that, although the cooperative action of P-ATPases and K⁺ channels may account for some K⁺ accumulation, under extreme conditions more complex mechanisms must operate. In the case of the mycelial fungus, Neurospora crassa, and the giant pond alga, Chara australis, these mechanisms have been shown to be a K⁺/H⁺ symport and a K⁺/Na⁺ symport, respectively (Rodriguez-Navarro et al. 1986; Smith and Walker, 1989).

By a related argument, once gastric acid secretion had been shown to be the product of an ATPase (Ganser and Forte, 1973), it was clear that earlier ‘proofs’ of major intrinsic electrogenicity in the process must be spurious, since an ATP-driven pump that is known to create pH differences of ∼7 units or more has very little energy left for net movement of charge. Overall electroneutrality of the gastric H⁺-ATPase was quickly corroborated in ionophore experiments on vesicle-bound ATPase (Sachs et al. 1976), but almost a decade had passed before a convincing demonstration was assembled of the cooperating transport systems that gave the ensemble appearance of an electrogenic acid pump.

In fact, electrophysiological studies of the gastric P-ATPase attached to bilayer lipid membranes have recently shown the partial reaction driven by ATP to be electrogenic; i.e. charge displacement is an early step in proton pumping (van der Hijden et al. 1990). Actual release of the protons, however, requires entry of K⁺ into the enzyme, which compensates the displaced charge and, in the process, is itself countertransported. Because the supply of protons, ultimately from the blood, is effectively infinite, while the supply of potassium ions from the gastric surface is finite, continuation of proton secretion requires K⁺ to be recycled. This recycling is accomplished under normal circumstances by the opening of independent K⁺ and Cl⁻ channels in the luminal membrane of gastric parietal cells, resulting in transport of salt down a chemical gradient and providing Cl⁻ for secretion with H⁺ (Wolosin and Forte, 1981). The different processes are coordinated, at least in part, by the mode of gastric stimulation: insertion of intracellular tubulovesicles bearing the ATPase into apical canaliculi bearing the channels (Sachs et al. 1982).

As an introduction to the V-ATPases, Bill Harvey has already presented an excellent discussion of the way in which cooperating transport systems in the midgut of phytophagous insects can determine surprising practical functions for the ATPase. The original description of massive electrogenic K⁺ transport via goblet cells of the Cecropia midgut epithelium (Harvey et al. 1967, 1968), plus later identification of both a concentrated ATPase activity and a morphological feature - the so-called portasomes (Harvey et al. 1981) - which resemble the V(or F)-ATPases, evoked two different functional schemes: either this particular enzyme must differ from its likely congeners in actually pumping K⁺ rather than H⁺, or a bona fide H⁺-ATPase must be coordinated with other transport systems to give the ensemble appearance of a K⁺ pump. Only recently has the latter circumstance been clearly demonstrated (Wieczorek et al. 1989, 1991; Wieczorek, 1992; Harvey, 1992).