The devil is in the C-tail: a NBCe1-carbonic anhydrase bicarbonate transport metabolon?

Abstract

The concept of metabolons is well established in biochemistry as structural–metabolic complexes in cells that facilitate substrate channelling through the complex and is best exemplified by the citric acid cycle enzymes. Similarly, an arrangement of enzymes was suggested to promote the transport of acid/base equivalents across the plasma membrane by the Reithmeier group. The concept was supported by direct evidence of physical interaction of an acid/base transporter and carbonic anhydrase (Vince & Reithmeier, 1998) and emboldened by the demonstration of a functional advantage for such an arrangement called a ‘transport metabolon’ (Sterling et al. 2001). The direct protein interaction was first reported for the C-terminal of anion exchanger AE1 (band 3) and the cytosolic ubiquitous carbonic anhydrase II (CAII). Since then, the field has evolved to other acid/base transporters of the SLC4 gene family of HCO3− transporters, the Na+/H+ exchangers (NHE) and monocarboxylate transporters interacting with soluble or membrane-bound CA isozymes in various expression systems and native tissues. Discrepancies have since evolved both regarding the direct physical interaction of acid/base transporters and CA enzymes, and regarding the functional interaction between the individual molecules of such complexes. This ‘Perspectives’ article only addresses the case of the functional interaction of the human renal form of the electrogenic Na+:HCO3− cotransporter NBCe1-A (kNBCe1) and CAII when expressed in Xenopus laevis oocytes, but may prove highly relevant for the metabolon field as such. In one study, the NBCe1 function determined as the current slope conductance (Gm INBC) was not affected by injection of CAII protein or the expression of either NBCe1-CAII or eGFP-NBCe1-CAII fusion proteins (Lu et al. 2006). No effect of the cell-permeant CA inhibitor ethoxyzolamide (EZA) on the Gm INBC was observed. In a separate and opposing study, NBCe1 function assessed as the changes in membrane current (Im), the Na+ flux (d[Na+]i/dt), as well as the membrane conductance (Gm) was augmented by bovine CA protein injection or CAII co-expression (Becker & Deitmer 2007). The induced changes in current and Na+ fluxes were EZA-sensitive in this study. Thus, although the two studies applied similar methodological set-ups at first glance, the results were contrasting. Each paper is compelling to the reader, and both studies were carefully and elegantly performed. Moss and Boron (2020) first carefully replicate the findings by Lu et al. (2006) and then elegantly rule out differences in the amount of injected cRNA, CAII protein and eGFP tag as contributing factors to conflicting results by the two groups. Furthermore, they show that prolonged EZA exposure or the high EZA concentration by Lu et al. were not causing the inconsistency between the previous studies. It also appears that the variations in voltage-clamping protocols cannot explain the contradicting results per se (Moss & Boron 2020). Remarkably, the authors are able to closely replicate the fundamental observations by Becker & Deitmer (2007) that injection of bovine CA in oocytes expressing untagged NBCe1 increases the Na+ flux (d[Na+]i/dt)max as well as the membrane current Im, effects that are sensitive to CA inhibition by EZA. This is apparently only possible 1) in the absence of eGFP tag, 2) by bovine CA injection instead of human CAII, and 3) applying the prolonged -40mV voltage clamp and 10 s step duration of I-V ramps. Surprisingly, Moss and Boron demonstrate that injection of either human CAII or bovine CA alone in oocytes induces an electroneutral process that increases Na+ uptake and acid extrusion upon CO2-induced acid loading. The authors ascribe the effects to the induction of endogenous NHE activity, as this process is inhibited by EIPA. Interestingly, EIPA also prevents the EZA-sensitive change in Im and (d[Na+]i/dt)max induced by injection of bovine CA using the prolonged -40 mV voltage clamp protocol. Thus, Moss and Boron explain the discrepancy between the previous studies (Lu et al. 2006 vs. Becker & Deitmer 2007) by the induction of native NHE activity by exogenous CA in Xenopus oocytes. With the study by Moss and Boron (2020), the specific controversy regarding enhanced NBCe1 activity by CA in oocytes has most likely been fully resolved. The authors accomplished a systematic comparison of experimental approaches resulting in a study with high internal consistency presented in a respectful and transparent manner. The take-home message is that even the most careful studies by world-renowned laboratories can reach conflicting conclusions and that unexpected sources of discrepancies can be identified by hard and meticulous work. The findings by Moss and Boron (2020) should impact the broader transport metabolon field; however, the conclusions can probably not be extrapolated to all other cases of acid/base transporters, CA forms and cell types for which transport metabolons have been suggested. Hopefully, time will tell. None. Sole author. None.