Voltage coupling of primary H+ V-ATPases to secondary Na+- or K+-dependent transporters

Abstract

This review provides alternatives to two well established theories regarding membrane energization by H(+) V-ATPases. Firstly, we offer an alternative to the notion that the H(+) V-ATPase establishes a protonmotive force (pmf) across the membrane into which it is inserted. The term pmf, which was introduced by Peter Mitchell in 1961 in his chemiosmotic hypothesis for the synthesis of ATP by H(+) F-ATP synthases, has two parts, the electrical potential difference across the phosphorylating membrane, Deltapsi, and the pH difference between the bulk solutions on either side of the membrane, DeltapH. The DeltapH term implies three phases - a bulk fluid phase on the H(+) input side, the membrane phase and a bulk fluid phase on the H(+) output side. The Mitchell theory was applied to H(+) V-ATPases largely by analogy with H(+) F-ATP synthases operating in reverse as H(+) F-ATPases. We suggest an alternative, voltage coupling model. Our model for V-ATPases is based on Douglas B. Kell's 1979 'electrodic view' of ATP synthases in which two phases are added to the Mitchell model - an unstirred layer on the input side and another one on the output side of the membrane. In addition, we replace the notion that H(+) V-ATPases normally acidify the output bulk solution with the hypothesis, which we introduced in 1992, that the primary action of a H(+) V-ATPase is to charge the membrane capacitance and impose a Deltapsi across the membrane; the translocated hydrogen ions (H(+)s) are retained at the outer fluid-membrane interface by electrostatic attraction to the anions that were left behind. All subsequent events, including establishing pH differences in the outside bulk solution, are secondary. Using the surface of an electrode as a model, Kell's 'electrodic view' has five phases - the outer bulk fluid phase, an outer fluid-membrane interface, the membrane phase, an inner fluid-membrane interface and the inner bulk fluid phase. Light flash, H(+) releasing and binding experiments and other evidence provide convincing support for Kell's electrodic view yet Mitchell's chemiosmotic theory is the one that is accepted by most bioenergetics experts today. First we discuss the interaction between H(+) V-ATPase and the K(+)/2H(+) antiporter that forms the caterpillar K(+) pump, and use the Kell electrodic view to explain how the H(+)s at the outer fluid-membrane interface can drive two H(+) from lumen to cell and one K(+) from cell to lumen via the antiporter even though the pH in the bulk fluid of the lumen is highly alkaline. Exchange of outer bulk fluid K(+) (or Na(+)) with outer interface H(+) in conjunction with (K(+) or Na(+))/2H(+) antiport, transforms the hydrogen ion electrochemical potential difference, mu(H), to a K(+) electrochemical potential difference, mu(K) or a Na(+) electrochemical potential difference, mu(Na). The mu(K) or mu(Na) drives K(+)- or Na(+)-coupled nutrient amino acid transporters (NATs), such as KAAT1 (K(+) amino acid transporter 1), which moves Na(+) and an amino acid into the cell with no H(+)s involved. Examples in which the voltage coupling model is used to interpret ion and amino acid transport in caterpillar and larval mosquito midgut are discussed.

Fig. 1.

H⁺ V-ATPase generates membrane potential. A H⁺ V-ATPase (V) is inserted into an ideal lipid bilayer (M) of a membrane; upon hydrolysis of ATP in the inside bulk fluid (R), H⁺ is translocated across the bilayer (M) to the fluid membrane interface (SL) and is separated from its gegenion, A^–, which remains at the inner fluid membrane interface (SR). H⁺ is held at the fluid membrane interface (SL) by electrostatic attraction to its gegenion. A membrane potential is generated with the outside positive (+) to the inside (–).

Fig. 2.

H⁺ is replaced at the fluid membrane interface by Na⁺. If the outside bulk solution contains, say, NaCl at a concentration of, say, 10 mmol l^–1 and the H⁺ concentration is, say, 10^–4 mmol l^–1 (pH 7) there would be 100,000 Na⁺s for every H⁺ in the outside bulk fluid, so H⁺ at the fluid membrane interface would move into the outside bulk phase, being replaced at the interface by Na⁺ and the outside bulk fluid would become acidic to the extent limited by the capacitance of the membrane.

Fig. 3.

V_m drives a K⁺ or Na⁺/2H⁺ antiporter. The membrane potential (V_m) established by the H⁺ V-ATPase drives two H⁺ into the cell and one Na⁺ out to the fluid membrane interface via a K⁺ or Na⁺/2H⁺ antiporter (A). The at the interface is replaced by . The voltage is changed but little and a steady state is established in which H⁺ can recycle and Na⁺ can move out of the cells and alkalinize the lumen as long as there is a K⁺ or Na⁺ salt and ATP in the inside bulk fluid. The pH of the outside bulk fluid changes from <6.0 to >10.5 as H₂CO₃ is converted to K₂CO₃ or Na₂CO₃.

Fig. 4.

V_m also drives Na⁺ coupled amino acid symport. V_m drives Na⁺ that is stoichiometrically linked to an amino acid into the cell via a nutrient amino acid transporter (NAT, N). Although the membrane voltage is little changed, Na⁺ can recycle and amino acids can move into the cell as long as there is a sodium salt and an amino acid with affinity for the NAT in the outside bulk fluid. Although the energy for the symport process is ATP hydrolysis by the H⁺ V-ATPase there is no H⁺ involved in the symport per se, which is driven by the Na⁺ electrochemical potential difference

Fig. 5.

Diagram of transverse section through the posterior midgut of fifth instar Manduca sexta larva showing two columnar cells enclosing a goblet cell [modified from Cioffi and Wolfersberger (Cioffi and Wolfersberger, 1983)]. pm, peritrophic membrane; CCAM, columnar cell apical membrane; LM, lateral membrane; GCAM, goblet cell apical membrane; BM, basal membrane; MV, microvilli; M, mitochondrion; SJ, septate junction; GC, goblet cavity; AMP, apical membrane projection; P, portasome (equivalent to V₁ sector of H⁺ V-ATPase); BI, basal infolding; BL, basal lamina. The region in the small square is enlarged in C showing the CCAM with nutrient amino acid transporter (N) inserted into the membrane of a microvillus (equivalent to the BBM). The region in the large square is enlarged in B showing the GCAM with portasomes (V₁ ATPase sectors) as round black dots with key thermodynamic parameters for the epithelium. Thermodynamic data for the electrical potential and chemical concentration differences (Dow and Peacock, 1989; Dow, 1992; Dow, 1984) were combined by Dow (Dow, 1992) into a revised view of pH and ion regulation in the caterpillar midgut that includes the H⁺ V-ATPase and K⁺/2H⁺ antiporter concept. Dow's model is combined with Cioffi's diagram of the ultrastructure of the anterior midgut epithelium (Cioffi and Wolfersberger, 1983) to describe the pathway by which K⁺ is translocated from the hemolymph to the goblet cell cytoplasm, then to the goblet cavity, and finally through the goblet valve to the lumen. The relevant point here is that the force which drives H⁺ from the goblet cavity back into the cell via the K⁺/2H⁺ antiporter is the 269 Δψ across the GCAM that was generated by the H⁺ V-ATPase. The antiport results in a [K⁺] of 190 mmol l^–1 in the cavity compared with a [K⁺] of 130 mmol l^–1 in the cell while the cavity pH is rendered slightly more alkaline than that of the cells (Chao et al., 1991). The sulfate groups projecting from the GCAM into the goblet cavity were deduced from X-ray microanalysis data (Dow et al., 1984). They provide strong anions so that the predominant ions in the cavity are 2K⁺ and SO₄^2–. When K⁺ passes through the goblet valve into the lumen the predominant anion there is carbonate and the 2K⁺ CO₃^2– accounts for the high lumen pH of 11. This route is difficult to envision in terms of Mitchell's protonmotive force, three-phase model but is predicted by the Kell and Harvey voltage coupled, five-phase model. Clearly it is the large membrane potential rather than the small pH difference (in the wrong direction) that is driving the K⁺/2H⁺ antiport across the GCAM.