Sharpey-Schafer lecture: gas channels

Abstract

The traditional dogma has been that all gases diffuse through all membranes simply by dissolving in the lipid phase of the membrane. Although this mechanism may explain how most gases move through most membranes, it is now clear that some membranes have no demonstrable gas permeability, and that at least two families of membrane proteins, the aquaporins (AQPs) and the Rhesus (Rh) proteins, can each serve as pathways for the diffusion of both CO₂ and NH₃. The knockout of RhCG in the renal collecting duct leads to the predicted consequences in acid-base physiology, providing a clear-cut role for at least one gas channel in the normal physiology of mammals. In our laboratory, we have found that surface-pH (pH(S)) transients provide a sensitive approach for detecting CO₂ and NH₃ movement across the cell membranes of Xenopus oocytes. Using this approach, we have found that each tested AQP and Rh protein has its own characteristic CO₂/NH₃ permeability ratio, which provides the first demonstration of gas selectivity by a channel. Our preliminary AQP1 data suggest that all the NH₃ and less than half of the CO₂ move along with H₂O through the four monomeric aquapores. The majority of CO₂ takes an alternative route through AQP1, possibly the central pore at the four-fold axis of symmetry. Preliminary data with two Rh proteins, bacterial AmtB and human erythroid RhAG, suggest a similar story, with all the NH₃ moving through the three monomeric NH₃ pores and the CO₂ taking a separate route, perhaps the central pore at the three-fold axis of symmetry. The movement of different gases via different pathways is likely to underlie the gas selectivity that these channels exhibit.

Figure 1. Effect of extracellular NH₃/NH⁺₄ on intracellular pH (pH_i) of a squid giant axon, showing data (A), model of alkalinizing phase (B) and model of acidification during plateau phase (C)

Throughout the experiment in A, artificial seawater (ASW) with an extracellular pH of 7.70 flowed past a cannulated axon, which was suspended in a chamber of small volume. Intracellular pH and membrane potential (V_m) were monitored with glass microelectrodes. At the indicated times, the ASW was switched to one augmented with 10 mm NH₄Cl. Data are from Boron & De Weer (1976b). B shows that the influx of NH₃ leads to the consumption of intracellular H⁺ and thus a rise in pH_i. This process accounts for the rising phase of pH_i during the two NH₃/NH⁺₄ exposures in A. The influx of NH₃ in B leads to the dissociation of NH⁺₄ near the extracellular surface of the membrane. In the bulk (i.e. flowing) ASW, the NH₃/NH⁺₄ buffer was in equilibrium (NH₃+ H⁺⇌ NH⁺₄). C shows the system after NH₃ has equilibrated across the cell membrane; this equilibration corresponds to the peak pH_i during the second NH₃/NH⁺₄ pulse in A. After this equilibration, pH_i is dominated by the influx of NH⁺₄; this NH⁺₄ influx had been occurring since the beginning of the NH₃/NH⁺₄ exposure but its effect on pH_i had been overwhelmed by the influx of NH₃. Now, during the plateau phase, the influx of NH⁺₄ leads to a net dissociation of NH⁺₄ in the cytosol. This process accounts for the plateau-phase acidification (i.e. falling phase of pH_i) during the second NH₃/NH⁺₄ exposure in A. At the same time, the accumulation of NH₃ inside the cell now leads to the net efflux of NH₃, some of which consumes H⁺ on the outer surface of the cell, creating more NH⁺₄ (the NH₃/NH⁺₄ shuttle). Because the cell accumulated NH⁺₄ during the NH₃/NH⁺₄ exposure, the removal of extracellular NH₃/NH⁺₄ leads to a pH_i undershoot.

Figure 2. Effect of extracellular CO₂/HCO⁻₃ on intracellular pH of a squid giant axon, showing data (A), model of acidifying phase (B) and model of alkalinization during plateau phase (C)

Throughout the experiment in A, ASW with an extracellular pH of 7.70 flowed past a cannulated axon, which was suspended in a chamber of small volume. Intracellular pH and membrane potential were monitored with glass microelectrodes. During the indicated period, the ASW was switched to one equilibrated with 5% CO₂ and in which 50 mm NaHCO₃ replaced 50 mm NaCl. Data are from Boron & De Weer (1976b). B shows that the influx of CO₂ leads to the production of intracellular H⁺ and thus a fall in pH_i. This process accounts for the falling phase of pH_i during the CO₂/HCO⁻₃ exposure in A. The influx of CO₂ in B leads to the indicated reaction near the extracellular surface of the membrane. In the bulk (i.e. flowing) ASW, the CO₂/HCO⁻₃ buffer was in equilibrium (CO₂+ H₂O ⇌ H⁺+ HCO⁻₃). C shows the system after CO₂ has equilibrated across the cell membrane; this equilibration corresponds to the pH_i nadir during the CO₂/HCO⁻₃ pulse. After this equilibration, pH_i is dominated by ‘acid extrusion’, shown here as the active uptake of HCO⁻₃. This active uptake of HCO⁻₃ is mediated by a transporter called a Na⁺-driven Cl⁻–HCO⁻₃ exchanger (which may mediate uptake of CO₃²⁻ or NaCO₃⁻ ion pair). This HCO⁻₃ uptake had been occurring since the beginning of the CO₂/HCO⁻₃ exposure, but its effect on pH_i had been overwhelmed by the influx of CO₂. Now, during the plateau phase, HCO⁻₃ uptake leads to a consumption of H⁺ in the cytosol and thus the production of CO₂, leading to a net efflux of CO₂. This process accounts for the plateau-phase alkalinization (i.e. rising phase of pH_i) during the CO₂/HCO⁻₃ exposure in A. Because the cell accumulated HCO⁻₃ during the CO₂/HCO⁻₃ exposure, the removal of extracellular CO₂/HCO⁻₃ leads to a pH_i overshoot.

Figure 3. Effect of luminal versus basolateral NH₃/NH⁺₄ on intracellular pH of parietal cells of isolated, perfused gastric glands, with a luminal pH of 7.4 (A) and 8.0 (B)

Throughout the experiment, the lumen of the gland was perfused, and the basolateral surface (‘bath’) was superfused with CO₂/HCO⁻₃-free physiological saline at 37°C. Intracellular pH of multiple parietal and chief cells was measured using the pH-sensitive dye BCECF in conjunction with a digital-imaging system. Data are from Waisbren et al. (1994b); similar data were obtained on chief cells. During the indicated periods, either the luminal or the basolateral solution was switched to one in which 20 mm NH₄Cl replaced 20 mm NaCl. In A, both luminal and basolateral NH₃/NH⁺₄ solutions had a pH of 7.4. In B, the luminal NH₃/NH⁺₄ solution had a pH of 8.0 (and thus fourfold higher [NH₃]), whereas the basolateral NH₃/NH⁺₄ solution had a pH of 7.4. The basolateral NH₃/NH⁺₄ exposures produced pH_i transients (abcd) similar to that in the second NH₃/NH⁺₄ pulse in Fig. 1A, except that here the pH_i recovered from the acid load (de). However, the luminal exposures produced no significant pH_i changes. Together with other data, these observations showed that the apical membranes of parietal and chief cells have no detectable permeability to either NH₃ or NH⁺₄.

Figure 4. Effect of luminal versus basolateral CO₂/HCO⁻₃ on intracellular pH of parietal cells of isolated, perfused gastric glands, with a luminal pH of 7.4 (A) and 6.1 (B)

Throughout the experiment, the lumen of the gland was perfused, and the basolateral surface (‘bath’) was superfused with physiological saline at 37°C. Intracellular pH of multiple parietal and chief cells was measured using the pH-sensitive dye BCECF in conjunction with a digital-imaging system. Data are from Waisbren et al. (1994b); similar data were obtained on chief cells. During the indicated periods, either the luminal or the basolateral solution was switched to one equilibrated with CO₂. In A, both luminal and basolateral CO₂/HCO⁻₃ solutions had a pH of 7.4 achieved with 22 mm HCO⁻₃. The basolateral CO₂/HCO⁻₃ exposures produced pH_i transients (abcd) similar to that in Fig. 2A. However, the luminal exposure produced no significant pH_i changes. This experiment terminated with a nigericin calibration. In B, the luminal CO₂/HCO⁻₃ solution had a pH of 6.1 (with 100% CO₂/22 mm HCO⁻₃) but produced no significant pH_i change. The basolateral CO₂/HCO⁻₃ solutions had pH values of 7.4 (1% CO₂/4.4 mm HCO⁻₃ or 5% CO₂/22 mm HCO⁻₃) and produced the expected acidifications. Basolateral 200 μm DIDS blocked the pH_i recovery from the acid loads. Together with other data, these observations showed that the apical membranes of parietal and chief cells have no detectable permeability to either CO₂ or HCO⁻₃.

Figure 5. Effect of graded expression of human AQP1 on CO₂-induced acidification rate of Xenopus oocytes

Three oocytes (purple, orange and green records) injected with cRNA encoding human AQP1 were superfused with physiological saline at pH 7.5. Intracellular pH was monitored by impaling the cell with a liquid-membrane pH microelectrode and a conventional electrode for monitoring membrane potential. Data are from Cooper & Boron (1998). During the indicated periods, the extracellular solution was switched to one equilibrated with 1.5% CO₂/10 mm HCO⁻₃. The initial rate of pH_i decline is an index of the CO₂ permeability. After the electrophysiological recordings, the oocytes were dropped into deionized water and monitored for the time to lysis (shorter times correlating with greater osmotic water permeabilities). Together with other data, these observations showed that CO₂ can move through AQP1.

Figure 6. Model of surface pH (pH_S) changes caused by the influx of CO₂

The influx of CO₂ not only causes a fall of pH_i but also a transient rise of pH_S. The two inset pH_S records at the top right come from oocytes injected either with water or with cRNA encoding human AQP1, measured with liquid-membrane pH-sensitive microelectrodes that initially just touched the membrane surface and then were advanced an additional ∼40 μm. Data are from Musa-Aziz et al. (2009a).

Figure 7. Model of pH_S changes caused by the influx of NH₃

The influx of NH₃ not only causes a rise of pH_i but also a transient fall of pH_S. The two inset pH_S records at the top right come from oocytes injected either with water or with cRNA encoding human AQP1; the same oocytes as in Fig. 6. Data are from Musa-Aziz et al. (2009a).

Figure 8. Paired pH_S transients in single oocytes caused by the influx of CO₂ and then the influx of NH₃

Oocytes were injected with either water or cRNA encoding the indicated membrane protein, and then superfused with physiological saline at pH 7.5. The pH_S was monitored as outlined in Figs 6 and 7. For each oocyte, CO₂/HCO⁻₃ was introduced (left half of each panel), washed out (not shown), and then NH₃/NH⁺₄ was introduced (right half of each panel). A–C show that AQP1 but not three transporters can support the pH_S transients. D–H show that AQP1, AQP4, AQP5, AmtB and RhAG can each transport CO₂, but only AQP1, AmtB and RhAG can transport NH₃. Data are from Musa-Aziz et al. (2009a).

Figure 9. Mean channel-dependent changes in maximal rise of pH_S caused by CO₂ influx (A), maximal fall of pH_S caused by NH₃ influx (B) and osmotic water permeability (C)

The semi-quantitative index of maximal CO₂ flux,, is the maximal rise in pH_S (ΔpH_S) in oocytes expressing a channel, less the mean ΔpH_S of day-matched control oocytes (i.e. water-injected oocytes). The semi-quantitative index of maximal NH₃ flux, , is the greatest extent of the fall in pH_S (ΔpH_S) in oocytes expressing a channel, less the mean ΔpH_S of day-matched control oocytes (i.e. water-injected oocytes). The value of is not significantly different from zero for either AQP4 or AQP5. P_f* is the analogous figure for osmotic water permeability. Note that neither AmtB nor RhAG significantly conducted water. Data are from Musa-Aziz et al. (2009a).

Figure 10. Mean values ofandnormalized to P_f* (A) and mean values ofnormalized to(B)

The values in A were obtained by dividing the values of and for each oocyte by the value of P_f*. These ratios are semi-quantitative indices of CO₂/H₂O permeability ratios and the NH₃/H₂O permeability ratios. The values in B were obtained by dividing the values of for each oocyte by the value of . These ratios are semi-quantitative indices of CO₂/NH₃ permeability ratios. Since was not statistically different from zero for AQP4 and AQP5, the NH₃/H₂O ratios for these channels should not be different from zero. Likewise, the CO₂/NH₃ ratios are theoretically infinite. Data are from Musa-Aziz et al. (2009a).

Figure 11. Model of HCO⁻₃ reabsorption by the renal proximal tubule

Bicarbonate appears in the lumen of the proximal tubule as the result of glomerular filtration. Abbreviations: AQP1, aquaporin 1; CAII, carbonic anhydrase II; CAIV, carbonic anhydrase IV; NBCe1-A, renal splice variant of electrogenic Na/HCO₃ cotransporter 1; and NHE3, Na⁺–H⁺ exchanger 3.