In a recent study published in the journal Nature Communications, researchers structurally analyzed adenine nucleotide control and neurodegenerative diseases in ClC-3 exchangers.
Study: Structural basis of adenine nucleotides regulation and neurodegenerative pathology in ClC-3 exchanger. Image Credit: Lightspring / Shutterstock
ClC-3, a chloride/proton exchanger, is necessary for monitoring metabolic energy levels and is activated by adenosine triphosphate (ATP). Point-type mutations in the ClC-3 ion channels can cause neurological disorders in humans.
The cause of these mutations' gain of function remains unclear. ClC-3, ClC-4, and ClC-5 are metabolic energy sensors that aid in acidification and divert vacuolar-type ATPase (V-ATPase) electrical current. Knockout animals have severe retinal degeneration and aberrant protein breakdown.
Two disease-causing ClC-3 variants in humans, T570I and I607T generate higher current amplitudes at negatively charged transmembrane voltages, although the mechanism is unknown.
About the study
In the present study, researchers obtained high-resolution dimeric wild-type murine ClC-3 structures in apo states and combined them with adenosine monophosphate (AMP), adenosine diphosphate (ADP), and ATP. They also investigated the I607T mutation in apo and ATP-bound forms.
The researchers used cryogenic electron microscopy to identify ClC-3 structures under different circumstances, verifying the functional significance of pathway-lining residues using site-directed mutagenesis and patch-clamp recording. To facilitate ClC-3's plasma membrane localization, they inserted point mutations in the N terminus and altered E282 to Ala. They used surface biotinylation and confocal imaging to investigate the expression of ClC-3 mutants on cell surfaces.
Researchers assessed the effect of ATP on ClC-3 using the current ratio parameter, obtained by comparing the highest current amplitude achieved after ATP potentiation to the beginning amplitude before ATP potentiation. They hypothesized that ATP was a potentiator of this exchanger.
They examined the ClC-3 structure at 3.3 Å resolution to understand ATP binding processes and their potentiation. Observing an additional density resembling ATP between the two intracellular CBS domains, they injected ATP into ClC-3 protein samples before freezing to reduce hydrolysis.
Researchers performed all-atom molecular dynamic (MD) simulations of mClC-3ATP, mClC-3ADP, and mClC-3AMP to elucidate the binding configuration of ATP in ClC-3 exchangers. They also examined the pathways for ion transport inside the transmembrane domains and performed dynamic network analysis.
They introduced point mutations to the mutual information pathway, measuring ATP potentiation in these mutants, and they also resolved the structure of the I607T mutant in the apo and ATP-bound states.
Results
Concerning ClC-3 structure, researchers discovered chloride conducting channels in the transmembranous domains of every mClC-3apo subunit using CAVER plugins in PyMOL, which connects endosomal lumens to cytosols. Residues associated with ClC ion-conducting channels, like the E282 gating glutamate, are also found along this route, with the sidechain oriented upward.
The researchers discovered triple electron densities, most likely indicating three Cl- ions just below E282 (or Sext), proximal to Y630 (or Sin), and between the two (Scen), identical to those in the ClC-7 exchanger. E282 into Ala mutations decreased outward rectifications and enhanced current amplitude during hyperpolarization by uncoupling proton and chloride transport.
Mutating S453 at the Sext region to Arg demonstrated that the additional positive charges introduced likely promoted negatively charged chloride ion enrichment, increasing outward currents at positive-type membrane potentials.
The Y630A amino acid substitution increased current upon depolarization. The researchers discovered a split-ion route that begins proximal to the Scen region and runs behind Helix P and Helix O. E339, a conserved protonic glutamate present in ClC exchangers, was found in the cytosolic entry of this route. Mutating these residues increased ClC-3 currents.
The mClC-3 protein remained steady across the 300-ns simulation, with root mean square deviation (RMSD) plateauing at 30 ns. In comparison, AMP or ADP scarcely increased the current. Adding a saturating concentration of ADP or AMP (500 µM) resulted in electron densities in mClC-3ADP and mClC-3AMP structures.
ClC-3's ATP binding pocket showed a similar shape and sequence to ClC-5 and ClC-7, with ATP binding stabilized by residues from the N terminus, CBS1 domain, and CBS2 domain. Mutations on these residues resulted in much less current potentiation by ATP.
ATP interaction triggered conformational changes in primary residues in the transmembrane domains, enhancing Cl-ion transport. However, changes in conductance, open probabilities, and ClC-3 expressions may induce variations in Cl-ion transport in the mutants. Adding 10.0 mM ATP to patch pipette fluids in entire-cell patch configurations enhanced the exchanger's capacity to control ClC-3 through adenine nucleotides.
Conclusions
The study identified the structural basis for ClC-3 interactions with adenine nucleotides, elucidating its role as a metabolic energy sensor and linking cellular energy levels to endosomal activity. Researchers found Cl- and H+ ion transport pathways comparable to those observed in other ClC proteins and electron densities approximating Cl- ions and water molecules.
The S453R mutation in ClC-3 was associated with neurodevelopmental abnormalities. Adenine nucleotides regulate ClC-3 differently, with ATP binding to ClC-3 with greater affinity. The I607T mutation relates to myotonia congenita and early-onset neurodegeneration.
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