Proton channel of the chloroplast ATP synthase, CF0: Its time-averaged single-channel conductance as function of pH, temperature, isotopic and ionic medium composition

Abstract

The proton-driven ATP synthase of chloroplasts is composed of two elements, CFo and CFt. The membrane bound CFo conducts protons and the peripheral CFt interacts with nucleotides. By flash spectrophotometric techniques applied to thylakoid membranes from which about 50% of total CF~ was removed, we have previously determined the protonic (timeaveraged) single-channel conductance of CFo. Being in the order of 1 pS, it was sufficiently large to support the proposed role of CFo as a low-impedance access for protons to the coupling site in CFoCF1. On the other hand, it was too large to be readily reconciled with current concepts of proton supply to and proton conduction through the channel.We studied the time-averaged single-channel conductance of CFo under variation of pH, pD, ionic composition, temperature, and water/membrane structure with the following results:(i) CFo was proton-specific even against a background of 300 mM monovalent or 30 mM divalent cations. (ii) While the conductance of CFo was pH/pD-independent in the range from 5.6-8.0, in D20 it was lower by a constant factor of 1.7 than in H20. (iii) Addition of glycerol diminished the conductance and abolished the isotope effect. (iv) The Arrhenius activation energy was 42 kJ/mol and thus intermediate between the ones found for the water-filled pore, gramicidin (30 kJ/mol), and the mobile carrier, valinomycin (65 kJ/mol).The results implied that CFo is endowed with an extremely proton-specific (107-fold) selectivity filter. Its conductance is very high, and its conduction cycle is not necessarily rate limited by a protolytic reaction. The mechanisms of rapid proton supply to the channel mouth and of proton conduction remained enigmatic.

The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient.

“…Given the apparent pH-independence of the gH and the estimate of 10 fS for the unitary conductance of H § channels at pH 6, the unitary H + current at pHi 7.5 is evidently significantly larger than the diffusion-limited predictions above (0.1-0.23 fA). The F0 component of H+-ATPase also is a proton channel with a unitary conductance independent of pH over the range pH 5.6-8.0 (Althoff, Lill, and Junge, 1989;Wagner, Apley, and Hanke, 1989) and of similar magnitude, 10 fS (Schoenknecht, Junge, Lill, and Engelbrecht, 1986) up to 1 pS (Junge, 1989) depending on assumptions. Several mechanisms which were not included in the above calculations have been discussed in the context of attempts to explain how such large proton fluxes can be sustained by diffusion at physiological pH: local concentration of H § by charges on the membrane or channel, a large vestibule, buffering by membrane lipids, hydrolysis, or rapid surface conduction of protons (Haines, 1983;Nachliel and Gutman, 1984;Schulten and Schulten, 1985;Nagle and Dilley, 1986;Prats, Tocanne, and Teissie, 1987;Kasianowicz et al, 1987;Althoff et al, 1989;Junge, 1989;Wagner et al, 1989).…”

Proton conductance by the gramicidin water wire. Model for proton conductance in the F1F0 ATPases?

“…(c) Can a single water strand account for the proton selectivity observed in Fo? In chloroplasts, the permeability of CF0 to H+ is 107 times greater than Na+ permeability (Althoff et al, 1989). This could be due to selection at the channel (a sieve) or selection at an internal binding site.…”

“…The mechanism of proton flux in the Fo channel is not understood, but some of its characteristics are established: (a) the protonic current at maximum turnover rate is about 1200 H+ channel-' s-' in CFOCF, of intact chloroplasts (Althoff et al, 1989); (b) the Fo channel is highly selective to protons over other monovalent ions (H+/Na+ = 107 in CFo; Althoff et al, 1980); and (c) in Escherichia coli, mutations in Arg210 (Cain and Simoni, 1989), His245 and Ser206 (Cain and Simoni, 1986) of the a subunit, and Asp61 of the c subunit (Hoppe et al, 1982) block proton flux in Fo and are thought to be buried within the membrane. Nagle and Morowitz (1978) proposed that hydrogenbonded amino acid residues of membrane proteins might conduct protons by a hopping mechanism analogous to proton transport in ice and liquid water (Onsager, 1973).…”

The chaotropic anions thiocyanate and nitrate inhibit the electric current through the tonoplast ATPase of isolated vacuoles from suspension cells of Chenopodium rubrum

“…Chaotropics have also been reported to remove the coupling factor from F-ATPases, so that an electric and proton conductance, respectively, of the proteolipid can be observed in the mitochondrial (Schindler and Nelson 1982) as well as the chloroplast ATPase (Lill and Junge 1989, Althoff et al, 1989, Ltihring et al, 1989, Vacuolar ATPases in vivo face slightly chaotropic conditions, e,g,, by miilimolar cytoplasmic concentrations of nitrate (Miller and Smith 1992), or under cold conditions (Moriyama and Nelson 1989), We deemed it tiseful to test the interaction of chaotropic ions with the vacuolar ATPase on intact vactioies, rather than on vesicle preparations, to find out whether this interaction could be of physiological significance in vivo. Using the whole-vacuolar mode of the patch clamp technique, we measured the electric currents associated with proton pumping activity of the ATPase before and after the application of chaotropic ions on isolated vacuoles of Chenopodium rubrum suspension cells.…”