Dataset Information

Two Cl Ions and a Glu Compete for a Helix Cage in the CLC Proton/Cl- Antiporter.

ABSTRACT: The ubiquitously expressed CLC chloride transporters are involved in a great variety of physiological functions. The CLC protein fold is shared by Cl- channels and 2Cl-:1H+ antiporters. The antiporters pump three charges per cycle across the membrane with two Cl ions moving in the opposite direction of one proton. Multiconformational continuum electrostatics was used to calculate the coupled thermodynamics of the protonation of the extracellular-facing gating Glu (Ex) and Cl- binding to the external (Sx) and central (Sc) sites in CLC-ec1, the Escherichia coli exchanger. Sx, Sc, and Ex are buried within the protein where the intersection of two helix N-termini creates a region with a strong, localized positive potential for anion binding. Our chemical potential titrations describe the thermodynamic linkage for binding the Cl- to each site and protons to Ex. We find that the 2Cl-:1H+ binding stoichiometry is a result of Cl- binding to Sx requiring H+ binding to Ex, whereas Cl- binding to Sc does not lead to proton uptake. When Sx binds a Cl-, the protonated Ex moves upward, out of the positive helix cage. The increasing Ex proton affinity on binding the first Cl- reduces the cost of binding the second Cl- at either Sx or Sc. Despite the repulsion among the anions, the lowest energy states have two anions bound in the helix cage. The state with no Cl- is not favored electrostatically, but relies on Ex blocking Sx and on the central residues Y445 and S107 blocking Sc.

SUBMITTER: Chenal C

PROVIDER: S-EPMC5658741 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Publications

Two Cl Ions and a Glu Compete for a Helix Cage in the CLC Proton/Cl- Antiporter.

Chenal Cat C Gunner M R MR

Biophysical journal 20170901 5

The ubiquitously expressed CLC chloride transporters are involved in a great variety of physiological functions. The CLC protein fold is shared by Cl- channels and 2Cl-:1H+ antiporters. The antiporters pump three charges per cycle across the membrane with two Cl ions moving in the opposite direction of one proton. Multiconformational continuum electrostatics was used to calculate the coupled thermodynamics of the protonation of the extracellular-facing gating Glu ...[more]

PMID: 28877486

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Project description:Dent disease 1 (DD1) is a renal salt-wasting tubulopathy associated with mutations in the Cl-/H+ antiporter ClC-5. The disease typically manifests with proteinuria, hypercalciuria, nephrocalcinosis, and nephrolithiasis but is characterized by large phenotypic variability of no clear origin. Several DD1 cases have been reported lately with additional atypical hypokalemic metabolic alkalosis and hyperaldosteronism, symptoms usually associated with another renal disease termed Bartter syndrome (BS). Expression of the Bartter-like DD1 mutant ClC-5 G261E in HEK293T cells showed that it is retained in the ER and lacks the complex glycosylation typical for ClC-5 WT. Accordingly, the mutant abolished CLC ionic transport. Such phenotype is not unusual and is often observed also in DD1 ClC-5 mutants not associated with Bartter like phenotype. We noticed, therefore, that one type of BS is associated with mutations in the protein barttin that serves as an accessory subunit regulating the function and subcellular localization of ClC-K channels. The overlapping symptomatology of DD1 and BS, together with the homology between the proteins of the CLC family, led us to investigate whether barttin might also regulate ClC-5 transport. In HEK293T cells, we found that barttin cotransfection impairs the complex glycosylation and arrests ClC-5 in the endoplasmic reticulum. As barttin and ClC-5 are both expressed in the thin and thick ascending limbs of the Henle's loop and the collecting duct, interactions between the two proteins could potentially contribute to the phenotypic variability of DD1. Pathologic barttin mutants differentially regulated trafficking and processing of ClC-5, suggesting that the interaction between the two proteins might be relevant also for the pathophysiology of BS. Our findings show that barttin regulates the subcellular localization not only of kidney ClC-K channels but also of the ClC-5 transporter, and suggest that ClC-5 might potentially play a role not only in kidney proximal tubules but also in tubular kidney segments expressing barttin. In addition, they demonstrate that the spectrum of clinical, genetic and molecular pathophysiology investigation of DD1 should be extended.

Project description:We performed steered molecular dynamics (SMD) and umbrella sampling simulations of Cl- ion migration through the transmembrane domain of a prototypical E. coli CLC Cl-/H+ antiporter by employing combined quantum-mechanical (QM) and molecular-mechanical (MM) calculations. The SMD simulations revealed interesting conformational changes of the protein. While no large-amplitude motions of the protein were observed during pore opening, the side chain rotation of the protonated external gating residue Glu148 was found to be critical for full access of the channel entrance by Cl-. Moving the anion into the external binding site (Sext) induced small-amplitude shifting of the protein backbone at the N-terminal end of helix F. As Cl- traveled through the pore, rigid-body swinging motions of helix R separated it from helix D. Helix R returned to its original position once Cl- exited the channel. Population analysis based on polarized wavefunction from QM/MM calculations discovered significant (up to 20%) charge loss for Cl- along the ion translocation pathway inside the pore. The delocalized charge was redistributed onto the pore residues, especially the functional groups containing π bonds (e.g., the Tyr445 side chain), while the charges of the H atoms coordinating Cl- changed almost negligibly. Potentials of mean force computed from umbrella sampling at the QM/MM and MM levels both displayed barriers at the same locations near the pore entrance and exit. However, the QM/MM PMF showed higher barriers (~10 kcal/mol) than the MM PMF (~2 kcal/mol). Binding energy calculations indicated that the interactions between Cl- and certain pore residues were overestimated by the semi-empirical PM3 Hamiltonian and underestimated by the CHARMM36 force fields, both of which were employed in the umbrella sampling simulations. In particular, CHARMM36 underestimated binding interactions for the functional groups containing π bonds, missing the stabilizations of the Cl- ion due to electron delocalization. The results suggested that it is important to explore these quantum effects for accurate descriptions of the Cl- transport.

Dataset Information

Two Cl Ions and a Glu Compete for a Helix Cage in the CLC Proton/Cl- Antiporter.

Publications

Two Cl Ions and a Glu Compete for a Helix Cage in the CLC Proton/Cl<sup>-</sup> Antiporter.

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