Biology subjects

Fortea, E.

Publications and source records attributed to Fortea, E..

3 recordsLinked to original sources

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↗

bioRxiv2020

Transport mechanism of the neuronal excitatory amino acid transporter

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report Cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing for the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which conserved glutamate mediates proton symport.

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↗