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. 2010 Dec;95(12):1107-30.
doi: 10.1113/expphysiol.2010.055244. Epub 2010 Sep 17.

Sharpey-Schafer lecture: gas channels

Walter F Boron  1
Affiliations
Free PMC article

Sharpey-Schafer lecture: gas channels

Walter F Boron. Exp Physiol. 2010 Dec.
Free PMC article

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.

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Figures

Figure 1
Figure 1. Effect of extracellular NH3/NH+4 on intracellular pH (pHi) 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 (Vm) 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 pHi. This process accounts for the rising phase of pHi 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 pHi during the second NH3/NH+4 pulse in A. After this equilibration, pHi 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 pHi 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 pHi) 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 pHi undershoot.
Figure 2
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 pHi. This process accounts for the falling phase of pHi 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 pHi nadir during the CO2/HCO−3 pulse. After this equilibration, pHi 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 pHi 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 pHi) 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 pHi overshoot.
Figure 3
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 pHi transients (abcd) similar to that in the second NH3/NH+4 pulse in Fig. 1A, except that here the pHi recovered from the acid load (de). However, the luminal exposures produced no significant pHi 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
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 pHi transients (abcd) similar to that in Fig. 2A. However, the luminal exposure produced no significant pHi 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 pHi 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 pHi 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
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 pHi 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
Figure 6. Model of surface pH (pHS) changes caused by the influx of CO2
The influx of CO2 not only causes a fall of pHi but also a transient rise of pHS. The two inset pHS 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
Figure 7. Model of pHS changes caused by the influx of NH3
The influx of NH3 not only causes a rise of pHi but also a transient fall of pHS. The two inset pHS 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
Figure 8. Paired pHS 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 pHS 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 pHS 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
Figure 9. Mean channel-dependent changes in maximal rise of pHS caused by CO2 influx (A), maximal fall of pHS caused by NH3 influx (B) and osmotic water permeability (C)
The semi-quantitative index of maximal CO2 flux,formula image, is the maximal rise in pHS (ΔpHS) in oocytes expressing a channel, less the mean ΔpHS of day-matched control oocytes (i.e. water-injected oocytes). The semi-quantitative index of maximal NH3 flux, formula image, is the greatest extent of the fall in pHS (ΔpHS) in oocytes expressing a channel, less the mean ΔpHS of day-matched control oocytes (i.e. water-injected oocytes). The value of formula image is not significantly different from zero for either AQP4 or AQP5. Pf* 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
Figure 10. Mean values ofandnormalized to Pf* (A) and mean values ofnormalized to(B)
The values in A were obtained by dividing the values of formula image and formula image for each oocyte by the value of Pf*. 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 formula image for each oocyte by the value of formula image. These ratios are semi-quantitative indices of CO2/NH3 permeability ratios. Since formula image 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
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.
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