Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange

Reviewer #1 (Public Review):

Tippett et al present whole cell and proteoliposome transport data showing unequivocally that purified recombinant SLC26A6 reconstituted in proteoliposomes mediates electroneutral chloride/bicarbonate exchange, as well as coupled chloride/oxalate exchange unassociated with detectable current. Both functions contrast with the uncoupled chloride conductance mediated by SLC26A9. The authors also present a novel cryo-EM structure of full-length human SLC26A6 chloride/anion exchanger. As part of the structure, they offer the first partial view of the STAS domain previously predicted to be unstructured. They further define a single Arg residue of the SLC26A6 transmembrane domain required for coupled exchange, mutation of which yields apparently uncoupled electrogenic chloride transport mechanistically resembling that of SLC26A9, although of lower magnitude. The authors further apply to proteoliposomes for the first time a still novel approach to the measurement of bicarbonate transport using a bicarbonate-selective Europium fluorophor. The evidence strongly supports the authors' claims and conclusions, with one exception.

The manuscript has numerous strengths.

As a structural biology contribution, the authors extend the range of SLC26 structures to SLC26A6, comparing it in considerable detail to the published SLC26A9 structure, and presenting for the first time the structure of a portion of the STAS IVS domain of SLC26A6 long considered unstructured.

The authors also apply a remarkably extensive range of creative technical approaches to assess the functional mechanisms of anion transport by SLC26A6, among them the first application of the novel, specific bicarbonate sensor Eu-L1+ to directly assess bicarbonate transport in reconstituted proteoliposomes. The authors also present the first (to this reviewer's knowledge) functional proteoliposome reconstitution of chloride-bicarbonate exchange mediated by an SLC26 protein. They define a residue in surrounding the anion binding pocket which explains part of the difference in anion exchange coupling between SLC26A6 and SLC26A9. In the setting of past conflicting results, the current work also contributes to the weight of previous evidence demonstrating that SLC26A6 mediates electroneutral rather than electrogenic Cl-/HCO3- exchange.

Each of these achievements constitutes a significant advance in our understanding.

The paper has only a few weaknesses. One is an incomplete explanation of the mechanistic determinants of anion exchange coupling in SLC26A6 vs. uncoupled anion transport by SLC26A9. A second minor weakness is the inconsistently repeated conclusion that SLC26A6 mediates strictly coupled chloride/oxalate exchange. The data presented do not measure the stoichiometry of Cl-/oxalate exchange. The AMCA proteoliposome assay documented extracellular oxalate-dependent proteoliposomal anion transport that was most simply interpreted as coupled exchange, whereas no stoichiometric coupled exchange was documented in the AMCA experiments as presented.

Overall, the manuscript represents an important advance in our understanding of the SLC26 protein family and of coupled vs uncoupled carrier-mediated anion transport.

Reviewer #2 (Public Review):

The eleven paralogs of SLC26 proteins in humans exhibit a remarkable range of functional diversity, spanning from slow anion exchangers and fast anion transporters with channel-like properties, to motor proteins found in the cochlear outer hair cells. In this study, the authors investigate human SLC26A6, which functions as a bicarbonate (HCO3-)/chloride (Cl-) and oxalate (C2O42-)/Cl- exchanger, combining cryo-electron microscopy, electrophysiology, and in vitro transport assays. The authors provide compelling evidence to support the idea that SLC26A6's exchange anions at equimolar stoichiometry, leading to the electroneutral and electrogenic transport of HCO3-/ Cl- and C2O42-/Cl-, respectively. Furthermore, the structure of SLC26A6 reveals a close resemblance to the fast, uncoupled Cl- transporter SLC26A9, with the major structural differences observed within the anion binding site. By characterizing an amino acid substitution within the SLC26A6 anion binding site (R404V), the authors also show that the size and charge variance of the binding pocket between the two paralogs could, in part, contribute to the differences in their transport mechanisms.

The strength of this work lies in the reductionist, in vitro approach that the authors took to characterize the transport process of SLC26A6. The authors used and developed an array of functional experiments, including two electrogenic transport assays - a fast kinetic (electrophysiology) and a slow-kinetic (fluorescent-based ACMA) - and two electroneutral transport assays, probing for Cl- (lucigenin) and HCO3- (europium), which are well executed and characterized. The structural data is also of high quality and is the first structure of an SLC26 coupled anion exchanger, providing essential information for clarifying our understanding of the functional diversity between the SLC26 family of proteins.

To my knowledge, the outward-facing conformational state has not been determined for any mammalian SLC26 paralog, which limits the mechanistic interpretation of transport and is a weaker point of this manuscript. However, this is a very minor point.

Reviewer #3 (Public Review):

The mechanistically diverse SLC26 transporters play a variety of physiological roles. The current manuscript establishes the SLC26A6 subtype as electroneutral chloride/bicarbonate exchanges and reports its high-resolution structure with chloride bound.

The claims in this manuscript are all well-supported by the data. Strengths include the comprehensive functional analysis of SLC26A6 in reconstituted liposome vesicles. The authors employ an array of assays, including chloride sensors, a newly developed fluorescent probe for bicarbonate, and assays to detect the electrogenicity of anion exchange. With this assortment of assays, the authors are able to establish the anion selectivity and stoichiometry of SLC26A6. Another strength of the manuscript is the functional comparison with SLC26A9, which permits fast, passive chloride transport, in order to benchmark the SLC26A6 activity. The structural analysis, including the assignment of the chloride binding site, is also convincing. The structural details and the chloride binding site are well-conserved among SLC26s. Finally, the authors present an interesting discussion comparing the structures of SLC26A5, SLC26A6, and SLC26A9, and how the details of the chloride binding site might influence the mechanistic distinctions between these similar transporters.