The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.

Extra-virgin olive oil (EVOO) is a critical component of the Mediterranean diet, which has been found beneficial to human health. Bitterness is often positively associated with the presence of phenolic compounds in EVOO. There are twenty-five bitter taste receptors (TAS2Rs) in humans, each of which responds to specific bitter tastants. The identity of phenolic compounds and the bitter taste receptors they stimulate remain unknown. In this study, we isolated 12 phenolic and secoiridoid compounds from the olive fruit and the oil extracted from it, and tested their ability to stimulate bitter taste receptor activity, using a calcium mobilization functional assay. Our results showed that seven out of twelve studied compounds activated TAS2R8, and five of them activated TAS2R1, TAS2R8, and TAS2R14. The phenolic compounds oleuropein aglycon and ligstroside aglycon were the most potent bitter tastants in olive oil. TAS2R1 and TAS2R8 were the major bitter taste receptors activated most potently by these phenolic compounds. The results obtained here could be utilized to predict and control the bitterness of olive oil based on the concentration of specific bitter phenolics produced during the milling process of olives.

Significance: Cholesterol is one of the main components found in plasma membranes and is involved in lipid-dependent signaling enabled by integral membrane proteins such as inwardly rectifying potassium (Kir) channels. Similar to other ion channels, most of the Kir channels are down-regulated by cholesterol. One of the very few notable exceptions is Kir3.4, which is up-regulated by this important lipid. Here, we discovered and characterized a molecular switch that controls the impact (up-regulation vs. down-regulation) of cholesterol on Kir3.4. Our results provide a detailed molecular mechanism of tunable cholesterol regulation of a potassium channel.

The Na-K-2Cl cotransporter-1 (NKCC1) is an electroneutral Na ⁺ -dependent transporter responsible for simultaneously translocating Na ⁺ , K ⁺ , and Cl ⁻ ions into cells. In human tissue, NKCC1 plays a critical role in regulating cytoplasmic volume, fluid intake, chloride homeostasis, and cell polarity. Here, we report four structures of human NKCC1 (hNKCC1), both in the absence and presence of loop diuretic (bumetanide or furosemide), using single-particle cryo–electron microscopy. These structures allow us to directly observe various novel conformations of the hNKCC1 dimer. They also reveal two drug-binding sites located at the transmembrane and cytosolic carboxyl-terminal domains, respectively. Together, our findings enable us to delineate an inhibition mechanism that involves a coupled movement between the cytosolic and transmembrane domains of hNKCC1.

G protein–sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel–phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity.

Small-conductance Ca²⁺-activated K⁺ (K_Ca2.1-K_Ca2.3) channels modulate neuronal and cardiac excitability. We report cryo-electron microscopy structures of the K_Ca2.2 channel in complex with calmodulin and Ca²⁺, alone or bound to two small molecule inhibitors, at 3.18, 3.50, 2.99 and 2.97 angstrom resolution, respectively. Extracellular S3-S4 loops in β-hairpin configuration form an outer canopy over the pore with an aromatic box at the canopy’s center. Each S3-S4 β-hairpin is tethered to the selectivity filter in the neighboring subunit by inter-subunit hydrogen bonds. This hydrogen bond network flips the aromatic residue (Tyr362) in the filter’s GYG signature by 180°, causing the outer selectivity filter to widen and water to enter the filter. Disruption of the tether by a mutation narrows the outer selectivity filter, realigns Tyr362 to the position seen in other K⁺ channels, and significantly increases unitary conductance. UCL1684, a mimetic of the bee venom peptide apamin, sits atop the canopy and occludes the opening in the aromatic box. AP14145, an analogue of a therapeutic for atrial fibrillation, binds in the central cavity below the selectivity filter and induces closure of the inner gate. These structures provide a basis for understanding the small unitary conductance and pharmacology of K_Ca2.x channels.