The Natural Compound Cinnamaldehyde is a Novel Activator of Calcium-Activated Chloride Channel.

Calcium-activated chloride channels (CaCCs) play important roles in a multitude of physiological processes, and in many cells types, TMEM16A was identified as the molecular basis of CaCC. Abnormal CaCC function has been implicated in variety of diseases, which reinforces the need for modulators of CaCCs/TMEM16A. However, there are few specific, clinical modulators of CaCCs. Here, we identified a potent novel activator of TMEM16A from a bank of traditional Chinese medicines (TCM) and explored its mechanism of activation by laser confocal scanning microscopy and patch clamping. Fluorescence data demonstrated that among the 36 tested TCM medicines, one compound, cinnamaldehyde (CA), can activate the TMEM16A channel in a dose-dependent manner. To determine the mechanism by which CA activates the TMEM16A channel, we performed an excised patch clamp experiment and measured the intracellular calcium concentration in fluorescence experiments. Our data show that CA activates TMEM16A channels by elevating the intracellular concentrations of calcium ions. The results of the whole-cell patch clamping showed that CA dose-dependently activates these channels, with an EC50 of 9.73 ± 5.64 µM at + 80 mV, and prolongs the deactivation of TMEM16A. Finally, we found that CA can strengthen contractions of the ileum in guinea pigs by activating TMEM16A. The results demonstrate that CA is a novel, natural activator of TMEM16A.

Hydrocinnamic Acid Inhibits the Currents of WT and SQT3 Syndrome-Related Mutants of Kir2.1 Channel.

Gain of function in mutations, D172N and E299V, of Kir2.1 will induce type III short QT syndrome. In our previous work, we had identified that a mixture of traditional Chinese medicine, styrax, is a blocker of Kir2.1. Here, we determined a monomer, hydrocinnamic acid (HA), as the effective component from 18 compounds of styrax. Our data show that HA can inhibit the currents of Kir2.1 channel in both excised inside-out and whole-cell patch with the IC50 of 5.21 ± 1.02 and 10.08 ± 0.46 mM, respectively. The time course of HA blockage and washout are 2.3 ± 0.6 and 10.5 ± 2.6 s in the excised inside-out patch. Moreover, HA can also abolish the currents of D172N and E299V with the IC50 of 6.66 ± 0.57 and 5.81 ± 1.10 mM for D172N and E299V, respectively. Molecular docking results determine that HA binds with Kir2.1 at K182, K185, and K188, which are phosphatidylinositol 4,5-bisphosphate (PIP2) binding residues. Our results indicate that HA competes with PIP2 to bind with Kir2.1 and inhibits the currents.

Two Ca(2+)-Binding Sites Cooperatively Couple Together in TMEM16A Channel.

TMEM16A is the molecular basis of calcium-activated chloride channels and shows Ca(2+)-dependent gating. It is critical to understand how the Ca(2+) sensors dynamically control the gate of TMEM16A. However, the detailed mechanism by which the calcium ions bind and open the channel is still obscure. In this study, the authors confirmed that there are two Ca(2+) sensors which cooperatively couple together in TMEM16A. Our data show that mutations at both Ca(2+)-sensitive domains, E447Y and E702Q-E705Q, weaken the Ca(2+) affinity for TMEM16A channel. The EC50 for WT, E447Y, and E702Q-E705Q are 0.53 ± 0.11, 14.5 ± 0.3, and 26.5 ± 3.6 µM, respectively. The triple mutation, including both of the Ca(2+) sensors, E447Y-E702Q-E705Q, with EC50 as 55.6 ± 5.1 µM, results in much further right-shifted dose response curve than the single sensor's mutations (E447Y, E702Q-E705Q) do, which indicates that there is a cooperation between the two Ca(2+)-sensitive domains. We also found that the divalent cations, both Ca(2+) and Sr(2+), share common mechanism of gating the TMEM16A.

Corydaline binds to a druggable pocket of hEAG1 channel and inhibits hepatic carcinoma cell viability.

Ether-à-go-go (EAG) potassium channels play a crucial role in the regulation of neuronal excitability and cancer progression, rendering them potential drug targets for cancer therapy. However, the scarcity of information regarding the selection sites on hEAG1 has posed a challenge in the discovery of new hEAG1 inhibitors. In this study, we introduced a novel natural product, corydaline, which selectively inhibits the hEAG1 channel without sensitivity to other KCNH channels. The IC50 of corydaline for the hEAG1 channel was 11.3 ± 0.6 µM, whereas the IC50 for hEAG2 and hERG1 were 73.6 ± 9.9 µM and 111.4 ± 8.5 µM, respectively. Molecular dynamics simulations together with site-directed mutagenesis, have unveiled that the site corydaline forms interactions with Lys217, Phe273, Pro276, Trp295 and Arg366, situated within the intracellular transmembrane segments S1-S4 of the voltage-sensor domain, be considered a novel drug pocket for hEAG1. Additionally, the intergaration of sequence alignment and 3D structural modeling revealed differences between the voltage sensor domain of hEAG1 channel and other EAG channels, suggesting the feasibility of a VSD modulation approach that could potentially lead to the selective inhibition of hEAG1 channels. Furthermore, antitumor experiments demonstrated that corydaline can inhibit the proliferation and migration of hepatic carcinoma cells by targeting hEAG1. The identification of this novel druggable pocket offers the possibility for drug screening against diseases linked to abnormal hEAG1 channels.

Combining fragment homology modeling with molecular dynamics aims at prediction of Ca²âº binding sites in CaBPs.

The family of calcium-binding proteins (CaBPs) consists of dozens of members and contributes to all aspects of the cell's function, from homeostasis to learning and memory. However, the Ca²âº-binding mechanism is still unclear for most of CaBPs. To identify the Ca²âº-binding sites of CaBPs, this study presented a computational approach which combined the fragment homology modeling with molecular dynamics simulation. For validation, we performed a two-step strategy as follows: first, the approach is used to identify the Ca²âº-binding sites of CaBPs, which have the EF-hand Ca²âº-binding site and the detailed binding mechanism. To accomplish this, eighteen crystal structures of CaBPs with 49 Ca²âº-binding sites are selected to be analyzed including calmodulin. The computational method identified 43 from 49 Ca²âº-binding sites. Second, we performed the approach to large-conductance Ca²âº-activated K⁺ (BK) channels which don't have clear Ca²âº-binding mechanism. The simulated results are consistent with the experimental data. The computational approach may shed some light on the identification of Ca²âº-binding sites in CaBPs.

Photothermal Conjugated Polymer Nanoparticles for Suppressing Breast Tumor Growth by Regulating TRPA1 Ion Channels.

Cancer cells survive by relying on oxidative stress defense against the accumulation of reactive oxygen species (ROS) during tumor formation. ROS-sensitive TRPA1 ion channels are overexpressed in breast cancer cells and induce a large influx of Ca2+ which upregulates the anti-apoptotic pathway to lead breast cancer cells to produce oxidative stress defense and enhance the resistance to ROS related chemotherapy. Targeting and inhibiting the TRPA1 ion channels are critical for breaking down the oxidative stress defense system and overcoming cellular resistance. Here, near-infrared (NIR) light-responsive conjugated polymer nanoparticles are designed and prepared to promote apoptosis of breast cancer cells, reduce cell drug resistance and suppress tumor growth through the remote and precise regulation of TRPA1 ion channels. Upon 808 nm laser irradiation, the nanoparticles block the formation of Ca2+ /CaM complex and regulate the content of MCL-1 protein. Especially, the nanoparticles overcome drug resistance of cancer cells, therefore accelerating apoptosis of cancer cells and suppressing tumor growth in mice. Compared with carboplatin, the volume of tumor induced by NPs-H decreases by 54.1%. This work provides a strategy to disrupt the oxidative stress defense system and downregulate the antiapoptotic signaling pathway in cancer cells.

Alleviating Neuroinflammation through Photothermal Conjugated Polymer Nanoparticles by Regulating Reactive Oxygen Species and Ca2+ Signaling.

Neuroinflammation is one of the important manifestations of the amyloid ß peptide (Aß) protein-induced neurotoxic signaling pathway in which the aggregation of Aß causes an increase in reactive oxygen species (ROS) and Ca2+ concentration. Here, near-infrared (NIR) photothermal-responsive conjugated polymer nanoparticles were designed to regulate ROS and Ca2+ signaling to alleviate neuroinflammation. Under 808 nm laser irradiation, the nanoparticles effectively penetrated the blood-brain barrier (BBB) and reduced the aggregation of Aß and partially disaggregated the aggregates outside the cell, thereby reducing ROS content which downregulated the oxidative stress damage to cells. Meanwhile, the nanoparticles reduced the concentration of Ca2+ by inhibiting the transient receptor potential melastatin-related 2 (TRPM2) ion channel inside the cell. Ultimately, the concentration of inflammatory factor tumor necrosis factor-α was decreased. This study provides an effective strategy to reduce neuroinflammation by simultaneously regulating ROS and Ca2+ signaling.

Molecular dynamics simulation of TMEM16A channel: Linking structure with gating.

TMEM16A, the calcium-activated chloride channel, is broadly expressed and plays pivotal roles in diverse physiological processes. To understand the structural and functional relationships of TMEM16A, it is necessary to fully clarify the structural basis of the gating of the TMEM16A channel. Herein, we performed the protein electrostatic analysis and molecular dynamics simulation on the TMEM16A in the presence and absence of Ca2+. Data showed that the separation of TM4 and TM6 causes pore expansion, and Q646 may be a key residue for the formation of π-helix in the middle segment of TM6. Moreover, E705 was found to form a group of H-bond interactions with D554/K588/K645 below the hydrophobic gate to stabilize the closed conformation of the pore in the Ca2+-free state. Interestingly, in the Ca2+ bound state, the E705 side chain swings 100o to serve as Ca2+-binding coordination and released K645. K645 is closer to the hydrophobic gate in the calcium-bound state, which facilitates the provision of electrostatic forces for chloride ions as the ions pass through the hydrophobic gate. Our findings provide the structural-based insights to understanding the mechanisms of gating of TMEM16A.

Styrax blocks inward and outward current of Kir2.1 channel.

Kir2.1 plays key roles in setting rest membrane potential and modulation of cell excitability. Mutations of Kir2.1, such as D172N or E299V, inducing gain-of-function, can cause type3 short QT syndrome (SQT3) due to the enlarged outward currents. So far, there is no clinical drug target to block the currents of Kir2.1. Here, we identified a novel blocker of Kir2.1, styrax, which is a kind of natural compound selected from traditional Chinese medicine. Our data show that styrax can abolish the inward and outward currents of Kir2.1. The IC50 of styrax for WT, D172N and E299V are 0.0113 ± 0.00075, 0.0204 ± 0.0048 and 0.0122 ± 0.0012 (in volume), respectively. The results indicate that styrax can serve as a novel blocker for Kir2.1.

TMEM16A/B associated CaCC: structural and functional insights.

Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Transmembrane proteins 16A and 16B (TMEM16A/B) were identified to be the best molecular identities of CaCCs to date. This makes molecular investigation of CaCCs become possible. This review discusses the latest findings of TMEM16A/B associated CaCCs, the calcium and voltage dual dependence，the reorganization of Ca(2+)-binding site, the mechanisms of direct or indirect activation, the structure-functional relationship, and the possible stereoscopic structure. TMEM16A and other members of the family are associated with several kinds of cancers and other chloride channelopathies. An understanding of TMEM16 associated channel proteins will shed some light on their role in oncology and in pharmacology development.