Evaluating the role of carbonic anhydrases in the transport of HCO3--related species

Abstract

The soluble enzyme carbonic anhydrase II (CAII) plays an important role in CO(2) influx and efflux by red blood cells (RBCs), a process initiated by changes in the extracellular [CO(2)] (CO(2)-initiated CO(2) transport). Evidence suggests that CAII may be part of a macromolecular complex at the inner surface of the RBC membrane. Some have suggested CAII specifically binds to a motif on the cytoplasmic C terminus (Ct) of the Cl-HCO(3) exchanger AE1 and some other members of the SLC4 family of HCO(3)(-) transporters, a transport metabolon. Moreover, others have suggested that this bound CAII enhances the transport of HCO(3)(-)-related species-HCO(3)(-), CO(3)(), or CO(3)() ion pairs-when the process is initiated by altering the activity of the transporter (HCO(3)(-)-initiated HCO(3)(-) transport). In this review, I assess the theoretical roles of CAs in the transport of CO(2) and HCO(3)(-)-related species, concluding that although the effect of bound CAII on CO(2)-initiated CO(2) transport is expected to be substantial, the effect of bound CAs on HCO(3)(-)-initiated HCO(3)(-) transport is expected to be modest at best. I also assess the experimental evidence for CAII binding to AE1 and other transporters, and the effects of this binding on HCO(3)(-)-initiated HCO(3)(-) transport. The early conclusion that CAII binds to the Ct of AE1 appears to be the result of unpredictable effects of GST in the GST fusion proteins used in the studies. The early conclusion that bound CAII speeds HCO(3)(-)-initiated HCO(3)(-) transport appears to be the result of CAII accelerating the pH changes used as a read-out of transport. Thus, it appears that CAII does not bind directly to AE1 or other SLC4 proteins, and that bound CAII does not substantially accelerate HCO(3)(-)-initiated HCO(3)(-) transport.

Fig. 1

Predicted effect of intracellular CAII on CO₂ influx into red blood cells. (A) Equilibrium before a change in [CO₂]. (B) Increase in extracellular [CO₂] from 1.20 to 1.40 mM. (C) CO₂ influx leads to an increase in intracellular [CO₂]. The specific value of 1.35 mM is for illustration only. Up until this point, we assume that CAII is inactive. (D) Hypothetical sudden activation of CAII causes the conversion of CO₂ to ${\mathrm{HCO}}_3^{-}$. Note that, although the fractional rise in ${\left[{\mathrm{HCO}}_3^{-}\right]}_{\mathrm{i}}$ is the same as the fractional rise in [CO₂]_i, the absolute rise in ${\left[{\mathrm{HCO}}_3^{-}\right]}_{\mathrm{i}}$ is an order of magnitude larger. Thus, we would expect ${\mathrm{HCO}}_3^{-}$ to contribute far more than CO₂ in the diffusion of “carbon” from the membrane into the center of the cell. Although not shown, non-${\mathrm{HCO}}_3^{-}$ buffers would contribute to the buffering of H⁺ in both the extra- and intracellular fluid.

Fig. 2

Predicted effect of intracellular CAII on ${\mathrm{HCO}}_3^{-}$-initiated ${\mathrm{HCO}}_3^{-}$ influx into red blood cells. (A) Equilibrium before a change in [CO₂]. (B) Removal of extracellular Cl⁻ promotes sudden activation of transport by the Cl–HCO₃ exchanger AE1. (C) ${\mathrm{HCO}}_3^{-}$ influx leads to an increase in intracellular $\left[{\mathrm{HCO}}_3^{-}\right]$. The specific value of 13 mM is for illustration only. Up until this point, we assume that CAII is inactive. (D) Hypothetical sudden activation of CAII causes the conversion of ${\mathrm{HCO}}_3^{-}$ to CO₂. Note that, although the fractional rise in ${\left[{\mathrm{HCO}}_3^{-}\right]}_{\mathrm{i}}$ is the same as the fractional rise in [CO₂]_i, the absolute rise in ${\left[{\mathrm{HCO}}_3^{-}\right]}_{\mathrm{i}}$ is an order of magnitude larger. Although not shown, non-${\mathrm{HCO}}_3^{-}$ buffers would contribute to the buffering of H⁺ in both the extra- and intracellular fluid.

Fig. 3

Predicted effect of extra- and intracellular CAs during the transport of ${\mathrm{HCO}}_3^{-}$ vs. ${\mathrm{CO}}_3^{=}$ vs. H⁺. (A) Hypothetical model of NBCe1 mediating the uptake of 1 Na⁺ and 2 ${\mathrm{HCO}}_3^{-}$. (B) Hypothetical model of NBCe1 mediating the uptake of 1 Na⁺ and 1 ${\mathrm{CO}}_3^{=}$. (C) Model of a Na-H exchanger mediating the uptake of 1 Na⁺ and the efflux of 1 H⁺. Although not shown, non-${\mathrm{HCO}}_3^{-}$ buffers would contribute to the buffering of H⁺ in both the extra- and intracellular fluid.

Fig. 4

Binding of liquid-phase GST-SLC4-Ct fusion proteins to immobilized CAII. (A) GST-AE1-Ct (red). (B) GST-NBCe1-Ct (green). (C) GST-NDCBE-Ct (blue). The inset schematizes the binding of GST-AE1-Ct to immobilized CAII. Each curve represents SLC4-specific binding, that is, the difference between binding of the GST-SLC4-Ct construct (probed with anti-GST and a secondary antibody) and the binding of GST to solid-phase CAII. These difference data were then fitted by a non-linear least-squares method to the Michaelis Menten equation. For GST-AE1-Ct, the apparent affinity (K_d) was 597 ± 186 nM and the maximal binding (B_max) was 0.88 ± 0.14 relative GST immunoreactivity units. For GST-NBCe1-Ct, K_d = 322 ±76 nM and B_max = 2.06 ± 0.20. For GST-NDCBE-Ct, K_d = 330 ± 165 nM and B_max = 0.60 ± 0.13. Data from ref [41].

Fig. 5

Binding of liquid-phase “pure” SLC4-Ct peptides (i.e., not fused to GST) to immobilized CAII. The insets schematize the binding of GST-AE1-Ct vs. AE1-Ct to immobilized CAII. In both cases, the bound material was detected with an antibody to the AE1-Ct (rather than with anti-GST, as in Fig. 4). Similar results were obtained with NBCe1-Ct (detected with anti-NBCe1-Ct) and His-tagged NDCBE-Ct (detected with anti-His). All values are relative to 1000 nM GST-AE1-Ct. Data from ref [41].

Fig. 6

Binding of liquid-phase CAII to immobilized GST, GST-AE1-Ct fusion protein, or “pure” AE1-Ct peptide (i.e., not fused to GST). The insets schematize the binding of CAII to immobilized GST, GST-AE1-Ct, or AE1-Ct. The bound CAII was detected with anti-CAII. Anti-AE1-Ct confirmed equal amounts of immobilized GST-AE1-Ct fusion protein and “pure” AE1-Ct peptides. Similar results were obtained with NBCe1 and NDCBE. All values are relative to 1000 nM GST-AE1-Ct. Data from ref. [41].

Fig. 7

Surface plasmon resonance (SPR) with immobilized CAII. Each panel shows an initial baseline, the wash-in of the liquid-phase “analyte,” and the washout of the analyte (A) Acetazolamide (ACZ) as the analyte. Increasing concentrations of ACZ (15, 62.5, and 250 nM) produced faster and increased levels of binding, fitted by a 1:1 Langmuir model. (B) Pure AE1-Ct peptide as the analyte. (C) Pure NBCe1-Ct peptide as the analyte. (D) Pure NDCBE-Ct peptide as the analyte. Responses in the last three panels are consistent with shifts in bulk refractive index (i.e., nonspecific interactions) caused by high peptide concentrations. The insets schematize the binding of ACZ, AE1-Ct, NBCe1-Ct, or NDCBE-Ct to immobilized CAII. Data from ref [41].

Fig. 8

Determination of the slope conductance of NBCe1-A. (A) Data are from a two-electrode voltage-clamp of a Xenopus oocyte expressing human NBCe1-A. The sequence of measurements was ND96 (HEPES-buffered, ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$-free), ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$ without inhibitor, ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$ with tenidap to block the transporter. (B) NBCe1 current. The symbols are the result of subtracting the green symbols in panel A from the red symbols. The dashed line is the result of a linear fit, which yielded a slope conductance of 14 μS. Data from ref [58].

Fig. 9

Effect of injected CAII and ethoxzolamide (EZA) on the slope conductance of NBCe1-A. (A) Comparison of the same oocytes, before and after injection of CAII+Tris (or just Tris). (B) Comparison of the same oocytes from panel A, before and after exposure to EZA. Measurements of pH_i (not shown) indicated that the injected CAII was catalytically active, and blocked by EZA. Data from ref [58].

Fig. 10

Effect of CAII—fused to the C terminus of the cotransporter—on the slope conductance of NBCe1-A. For the two bars at the left, the oocytes were expressing a construct consisting of GFP fused (via a 20aa linker) to human NBCe1-A, which was fused at its C terminus to human CAII. For the two bars at the right, the oocytes were expressing a construct consisting of GFP fused (via a 20aa linker) to human NBCe1-A, but without the CAII. Measurements of pH_i (not shown) indicated that the fused CAII was catalytically active. Data from ref [58].