Research Article: Structure of the human ClC-1 chloride channel

Date Published: April 25, 2019

Publisher: Public Library of Science

Author(s): Kaituo Wang, Sarah Spruce Preisler, Liying Zhang, Yanxiang Cui, Julie Winkel Missel, Christina Grønberg, Kamil Gotfryd, Erik Lindahl, Magnus Andersson, Kirstine Calloe, Pascal F. Egea, Dan Arne Klaerke, Michael Pusch, Per Amstrup Pedersen, Z. Hong Zhou, Pontus Gourdon, Raquel L. Lieberman

Abstract: ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue (“fast gate”) known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase (CBS) domains and the intracellular vestibule (“slow gating”). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1–related diseases.

Partial Text: CLC proteins comprise a large family of chloride (Cl−)-transporting integral membrane proteins with diverse physiological functions [1–3]. The first identified human member, ClC-1, is essential for maintaining the permeability of Cl− across the plasma membrane of skeletal muscle fibers, gCl, accounting for approximately 80% of the resting membrane conductance and assuring precise neuronal control of muscle contraction [3]. Mutations of the ClC-1 gene cause myotonia congenita, a disease that allows a single nerve action potential to trigger a series of muscle action potentials (myotonic runs), leading to prolonged muscle contraction [4–7].

Here, we have determined structures of full-length human ClC-1 using single-particle cryo-electron microscopy (cryo-EM), exploiting a purified protein sample that displays Cl−-dependent single-channel–derived ion conductance (S1 Fig and S1 Data). For structural characterization, sample in the presence of 100 mM Cl− at pH 7.5 and in the absence of nucleotides or antibodies was initially employed (Fig 1). Three-dimensional (3D) classification of particles resulted in several different groups, of which one yielded a 3.6 Å overall resolution density map for the transmembrane domain, allowing confident model building (S2–S4 Figs). The final model represents the membrane-spanning portion (note that the N terminus and intracellular αA helix are lacking) as well as parts of two C terminal’s so-called cystathionine-β-synthase (CBS) domains present per monomer (for which some cryo-EM density is left unmodeled) and includes several features that were not observed in the ClC-K structure (S5 Fig).

In summary, we report the molecular structure of Cl−-conducting human ClC-1, sharing an overall fold similar to other CLC proteins, with a narrow connecting pore and positively charged vestibules attracting Cl- ions similar to CFTR [38]. The structure exhibits several unique features, including shifts in the central GluGATE-TyrC pair, a more closed extracellular vestibule, and a wider penetration profile from the intracellular side, the latter representing a distinct feature of CLC channels separating them from transporters. We propose a model for adenine nucleotide and pH regulation of the common gate via CBS2 and the intracellular loops congruent with previous functional data. Overall, these findings significantly increase our understanding of Cl− conductance in physiology and open new opportunities for biomedicine. For example, the positively charged constriction of the extracellular vestibule and the putative 9-AC pocket may serve as favorable target sites for stimulators or inhibitors from outside or inside the cell, respectively.

Source:

http://doi.org/10.1371/journal.pbio.3000218