Structural Pharmacology of Cation-Chloride Cotransporters

Loop and thiazide diuretics have been cornerstones of clinical management of hypertension and fluid overload conditions for more than five decades. The hunt for their molecular targets led to the discovery of cation-chloride cotransporters (CCCs) that catalyze electroneutral movement of Cl− together with Na+ and/or K+. CCCs consist of two 1 Na+-1 K+-2 Cl− (NKCC1-2), one 1 Na+-1 Cl− (NCC), and four 1 K+-1 Cl− (KCC1-4) transporters in human. CCCs are fundamental in trans-epithelia ion secretion and absorption, homeostasis of intracellular Cl− concentration and cell volume, and regulation of neuronal excitability. Malfunction of NKCC2 and NCC leads to abnormal salt and water retention in the kidney and, consequently, imbalance in electrolytes and blood pressure. Mutations in KCC2 and KCC3 are associated with brain disorders due to impairments in regulation of excitability and possibly cell volume of neurons. A recent surge of structures of CCCs have defined their dimeric architecture, their ion binding sites, their conformational changes associated with ion translocation, and the mechanisms of action of loop diuretics and small molecule inhibitors. These breakthroughs now set the stage to expand CCC pharmacology beyond loop and thiazide diuretics, developing the next generation of diuretics with improved potency and specificity. Beyond drugging renal-specific CCCs, brain-penetrable therapeutics are sorely needed to target CCCs in the nervous system for the treatment of neurological disorders and psychiatric conditions

Single particle electron cryo-microscopy of a mammalian ion channel

The transient receptor potential (TRP) ion channel family is large and functionally diverse, second only to potassium channels. Despite their prominence within the animal kingdom, TRP channels have resisted crystallization and structural determination for many years. This barrier was recently broken when the three-dimensional structure of the vanilloid receptor 1 (TRPV1) was determined by single particle electron cryo-microscopy (cryo-EM). Moreover, this is the first example in which the near atomic resolution structure of an integral membrane protein was elucidated by this technique and in a manner not requiring crystals, demonstrating the transformative power of single particle cryo-EM for revealing high-resolution structures of integral membrane proteins, particularly those of mammalian origin. Here we summarize technical advances, in both biochemistry and cryo-EM, that led to this major breakthrough

Effects of Poncirin, a Citrus Flavonoid and Its Aglycone, Isosakuranetin, on the Gut Microbial Diversity and Metabolomics in Mice

Poncirin (PC) and its aglycone, isosakuranetin (IR), occur naturally in citrus fruits. This study aimed to explore the pathways behind the different health benefits of PC and IR by evaluating the effect of these two bioactive flavonoids on the gut microbial diversity and metabolomics of mice. The 16S rRNA gene sequencing was used to analyze the alteration of gut microbiota in mice after PC and IR intervention. The metabolic impact of PC and IR in mice were studied using a metabolomics approach based on LC-MS analysis. Results showed that, after 7 days intervention, PC and IR multiplied the abundance of Parabacteroides in mice’s intestinal tracts by 1.2 and 1.0 times, respectively. PC increased the abundance of Bacteroides by 2.4 times. IR reduced the Allobaculum abundance by 1.0 time and increased Alloprevotella abundance by 1.5 times. When mice were given PC, their fecal acetic acid level increased by 1.8 times, while their isobutyric and isovaleric acid content increased by 1.2 and 1.3 times, respectively. Supplementation with IR had no significant effect on the content of short-chain fatty acids (SCFAs) in the feces of mice. The potential urine biomarkers of mice in the PC group were involved in the digestion and absorption of protein and carbohydrate, as well as the metabolism of amino acids, such as glycine, serine, threonine, tryptophan, D-arginine, D-ornithine, etc. IR mainly affected the amino acid metabolic pathways in mice, including taurine and hypotaurine metabolism, glutathione metabolism, histidine metabolism, D-glutamate metabolism, etc. This study provided valuable clues for future research on the health promoting mechanisms of PC and IR

TRPV1 Channels Are Intrinsically Heat Sensitive and Negatively Regulated by Phosphoinositide Lipids

The capsaicin receptor, TRPV1, is regulated by phosphatidylinositol-4,5-bisphosphate (PIP(2)), although the precise nature of this effect (i.e., positive or negative) remains controversial. Here, we reconstitute purified TRPV1 into artificial liposomes, where it is gated robustly by capsaicin, protons, spider toxins, and, notably, heat, demonstrating intrinsic sensitivity of the channel to both chemical and thermal stimuli. TRPV1 is fully functional in the absence of phosphoinositides, arguing against their proposed obligatory role in channel activation. Rather, introduction of various phosphoinositides, including PIP(2), PI4P, and phosphatidylinositol, inhibits TRPV1, supporting a model whereby phosphoinositide turnover contributes to thermal hyperalgesia by disinhibiting the channel. Using an orthogonal chemical strategy, we show that association of the TRPV1 C terminus with the bilayer modulates channel gating, consistent with phylogenetic data implicating this domain as a key regulatory site for tuning stimulus sensitivity. Beyond TRPV1, these findings are relevant to understanding how membrane lipids modulate other "receptor-operated" TRP channels

Fusion of 2D CNN and 3D DenseNet for Dynamic Gesture Recognition

Gesture recognition has been applied in many fields as it is a natural human–computer communication method. However, recognition of dynamic gesture is still a challenging topic because of complex disturbance information and motion information. In this paper, we propose an effective dynamic gesture recognition method by fusing the prediction results of a two-dimensional (2D) motion representation convolution neural network (CNN) model and three-dimensional (3D) dense convolutional network (DenseNet) model. Firstly, to obtain a compact and discriminative gesture motion representation, the motion history image (MHI) and pseudo-coloring technique were employed to integrate the spatiotemporal motion sequences into a frame image, before being fed into a 2D CNN model for gesture classification. Next, the proposed 3D DenseNet model was used to extract spatiotemporal features directly from Red, Green, Blue (RGB) gesture videos. Finally, the prediction results of the proposed 2D and 3D deep models were blended together to boost recognition performance. The experimental results on two public datasets demonstrate the effectiveness of our proposed method