Structural basis of pH-dependent activation in a CLC transporter.

CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders.

Backbone amides are determinants of Cl selectivity in CLC ion channels.

Chloride homeostasis is regulated in all cellular compartments. CLC-type channels selectively transport Cl across biological membranes. It is proposed that side-chains of pore-lining residues determine Cl selectivity in CLC-type channels, but their spatial orientation and contributions to selectivity are not conserved.

Probing the Mechanism of Action of Cry41Aa on HepG2 through the Establishment of a Resistant Subline.

Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin.

TMEM16 scramblases thin the membrane to enable lipid scrambling.

TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. Scrambling was proposed to occur with lipid headgroups moving between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking.

A quantitative flux assay for the study of reconstituted Cl channels and transporters.

The recent deluge of high-resolution structural information on membrane proteins has not been accompanied by a comparable increase in our ability to functionally interrogate these proteins. Current functional assays often are not quantitative or are performed in conditions that significantly differ from those used in structural experiments, thus limiting the mechanistic correspondence between structural and functional experiments. A flux assay to determine quantitatively the functional properties of purified and reconstituted Cl channels and transporters in membranes of defined lipid compositions is described.

Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport.

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.

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

The CLC family comprises H-coupled exchangers and Cl channels, and mutations causing their dysfunction lead to genetic disorders. The CLC exchangers, unlike canonical 'ping-pong' antiporters, simultaneously bind and translocate substrates through partially congruent pathways. How ions of opposite charge bypass each other while moving through a shared pathway remains unknown.

Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes.

The four hyperpolarization-activated cylic-nucleotide gated (HCN) channel isoforms and their auxiliary subunit KCNE2 are important in the regulation of peripheral and central neuronal firing and the heartbeat. Disruption of their normal function has been implicated in cardiac arrhythmias, peripheral pain, and epilepsy. However, molecular details of the HCN-KCNE2 complexes are unknown.

The role of membrane-bound metal ions in toxicity of a human cancer cell-active pore-forming toxin Cry41Aa from Bacillus thuringiensis.

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts.

Cry6Aa1, a nematocidal and insecticidal toxin, forms pores in planar lipid bilayers at extremely low concentrations and without the need of proteolytic processing.

Cry6Aa1 is a () toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other toxins. Typical three-domain toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1-50 μg/ml range.