Identification of a crosstalk between ClC-1 C-terminal CBS domains and the transmembrane region.

CLC channels and transporters have large C-terminal regions which contain two cystathionine β-synthetase (CBS) domains. It has been hypothesized that conformational changes in these domains upon nucleotide binding modulate the gating of the CLC dimer. It is not clear how rearrangements that occur in the CBS domains are transmitted to the ion pathway, as CBS domains interact with the rest of the channel at multiple locations and some of these sites are not visible in recent solved cryogenic electron microscopy structures or are difficult to model using the AlphaFold server. Using ClC-1 as a model, we started working with a described ClC-1 mutation (H835R) located in the first alpha helix of the CBS2 domain which changes the voltage dependence of gating. We then identified several residues located in the disorganized loop after helix R (R-linker) that revert the phenotype of this mutation. We additionally proved that R-linker's function is connected to the CBS2 domain as current intensity, plasma membrane levels and gating defects of several R-linker variants were corrected by adding the mutation H835R. Furthermore, cross-linking studies using newly developed split-cysless ClC-1 channels containing specific cysteine mutants in the R-linker and the CBS2 domain indicate that these two regions are in close contact. Considering these new results, we propose that conformational changes occurring in the CBS domains could be transmitted to the CLC intracellular chloride binding site by means of its interaction with the R-linker. KEY POINTS: CBS domains, which are present as pairs in CLC proteins, are involved in the regulation by nucleotides of CLC gating. It is not clear how CBS domains interact with different regions of the transmembrane (TM) region to regulate gating. Using ClC-1 as a model, we investigated how a mutation in the second CBS domain dramatically changes the voltage dependence of gating taking advantage of recently solved structures and AlphaFold models of CLC proteins. Thus, we identified in the linker after helix R (R-linker) several revertant mutations of the gating defects caused by a mutation in the second CBS2 domain and vice versa, indicating that these two regions functionally interact. These interactions were biochemically proven by employing cysteine cross-linkings using a newly developed split-cysless ClC-1 channel. Based on these findings we experimentally prove that the R-linker is a key element for the transmission of the CBS conformational rearrangements to the TM region.