Leisle, L. | Search bioRxiv

bioRxiv2022

Backbone amides are key determinants of Cl- selectivity in CLC ion channels

Chloride homeostasis is tightly regulated in cellular compartments by dedicated channels and transporters. Whereas CLC-type channels select for Cl- over other anions, all other chloride channels are indiscriminate in their anionic permeability. Pore-lining side chains are thought to determine Cl- selectivity in CLC channels. However, orientation and functional roles of these side chains is not conserved among CLCs. All CLC pores are lined by backbone amides in a conserved structural arrangement, suggesting a role of mainchain groups in selectivity. We replaced pore-lining residues in the CLC-0 and bCLC-k channels with their respective -hydroxy acid counterparts using nonsense suppression method. This exchanges peptide-bond amides with ester-bond oxygens, incapable of hydrogen-bonding with permeating anions. Backbone substitutions functionally degrade inter-anion discrimination in a site-specific manner. These effects depend on the presence of a glutamate side chain that competes with ions permeating through the pore. Molecular dynamics simulations show that ion energetics within the bCLC-k pore are primarily determined by interactions with backbone amides. Insertion of an -hydroxy acid significantly alters ion selectivity and global pore hydration. We propose that backbone amides are conserved determinants of Cl- specificity in CLC channels in a mechanism reminiscent of that described for K+ channels.

biophysics↗

bioRxiv2019

Divergent Cl-and H + pathways underlie transport coupling and gating in CLC exchangers and channels

The CLC family of anion transporting proteins is comprised of secondary active H+-coupled exchangers and of Cl- channels. Both functional subtypes play key roles in human physiology, and mutations causing their dysfunction lead to numerous genetic disorders. Current models suggest that the CLC exchangers do not utilize a classical ping-pong mechanism of antiport, where the transporter sequentially interacts with one substrate at a time. Rather, in the CLC exchangers both substrates bind and translocate simultaneously while moving through partially congruent pathways. How ions of opposite electrical charge bypass each other while moving in opposite directions through a shared permeation pathway remains unknown. Here, we use MD simulations in combination with biochemical and electrophysiological measurements to identify a pair of highly conserved phenylalanine residues that form an aromatic pathway, separate from the Cl- pore, whose dynamic rearrangements enable H+ movement. Mutations of these aromatic residues impair H+ transport and voltage-dependent gating in the CLC exchangers. Remarkably, the role of the aromatic pathway is evolutionarily conserved in CLC channels. Using atomic-scale mutagenesis we show that the electrostatic properties and conformational flexibility of these aromatic residues are essential determinants of channel gating. Our results suggest that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters. We propose a unifying mechanism that describes the gating mechanism of CLC exchangers and channels.

biochemistry↗