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Pang C.-L.,Key Laboratory of Molecular Biophysics | Pang C.-L.,Hebei University of Technology | Yuan H.-B.,Key Laboratory of Molecular Biophysics | Yuan H.-B.,Hebei University of Technology | And 18 more authors.
Journal of Computer-Aided Molecular Design | Year: 2015

Calcium-activated chloride channels (CaCCs) play vital roles in a variety of physiological processes. Transmembrane protein 16A (TMEM16A) has been confirmed as the molecular counterpart of CaCCs which greatly pushes the molecular insights of CaCCs forward. However, the detailed mechanism of Ca2+ binding and activating the channel is still obscure. Here, we utilized a combination of computational and electrophysiological approaches to discern the molecular mechanism by which Ca2+ regulates the gating of TMEM16A channels. The simulation results show that the first intracellular loop serves as a Ca2+ binding site including D439, E444 and E447. The experimental results indicate that a novel residue, E447, plays key role in Ca2+ binding. Compared with WT TMEM16A, E447Y produces a 30-fold increase in EC50 of Ca2+ activation and leads to a 100-fold increase in Ca2+ concentrations that is needed to fully activate the channel. The following steered molecular dynamic (SMD) simulation data suggests that the mutations at 447 reduce the Ca2+ dissociation energy. Our results indicated that both the electrical property and the size of the side-chain at residue 447 have significant effects on Ca2+ dependent gating of TMEM16A. © 2015 Springer International Publishing Switzerland.

Xu J.-X.,Key Laboratory of Neural and Vascular Biology | Xu J.-X.,Hebei Medical University | Si M.,Key Laboratory of Neural and Vascular Biology | Si M.,Hebei Medical University | And 13 more authors.
Journal of Biological Chemistry | Year: 2014

Background: The mechanism and significance of phosphoinositide metabolism during heart stress stimulations are not clear. Results: Norepinephrine and angiotensin II increase cardiac phosphatidylinositol 4,5-bisphosphate levels via an enhanced interaction between phosphatidylinositol 4-kinase III and PKC, which correlate with a maintained systolic function. Conclusion: Cardiac phosphoinositide turnover is enhanced. Significance: A novel mechanism of phosphoinositide metabolism is described for modulation of cardiac function. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Guan B.,Key Laboratory of Neural and Vascular Biology | Guan B.,Key Laboratory of Pharmacology and Toxicology for New Drugs | Guan B.,Hebei Medical University | Chen X.,Key Laboratory of Neural and Vascular Biology | And 5 more authors.
Methods in Molecular Biology | Year: 2013

Two-electrode voltage clamp (TEVC) is a conventional electrophysiological technique used to artificially control the membrane potential (V m) of large cells to study the properties of electrogenic membrane proteins, especially ion channels. It makes use of two intracellular electrodes - a voltage electrode as V m sensor and a current electrode for current injection to adjust the V m, thus setting the membrane potential at desired values and recording the membrane current to analyze ion channel activities. Here we describe the use of TEVC in combination with exogenous mRNA expression in Xenopus oocytes for ion channel recording. © 2013 Springer Science+Business Media, LLC.

Chen X.,Key Laboratory of Neural and Vascular Biology | Chen X.,Hebei Medical University | Zhang X.,Key Laboratory of Neural and Vascular Biology | Zhang X.,Hebei Medical University | And 12 more authors.
Journal of Biological Chemistry | Year: 2011

In a previous study, we showed that membrane depolarization induced elevation of membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P 2, also known as PIP 2) and subsequently increased the KCNQ2/Q3 currents expressed in Xenopus oocytes through increased PI4 kinase activity. In this study, the underlying mechanism for this depolarization- induced enhancement of PIP 2 synthesis was further investigated. Our results indicate that activation of protein kinase C (PKC) isozyme βII was responsible for the enhanced PIP 2 synthesis. We found that phorbol-12-myristate, 13-acetate (PMA), an activator of PKC, mimicked the effects of the membrane depolarization by increasing KCNQ2/Q3 activity, elevating membrane PIP 2 levels and increasing activity of PI4 kinase β. Furthermore, membrane depolarization enhanced PKC activity. The effects of both depolarization and PMA were blocked by a PKC inhibitor or PI4 kinase β RNA interference. Further results demonstrate that the depolarization selectively activated the PKC βII isoform and enhanced its interaction with PI4 kinase β. These results reveal that the depolarization-induced elevation of membrane PIP 2 is through activation of PKC and the subsequent increased activity of PI4 kinase β. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Liu Y.,Key Laboratory of Neural and Vascular Biology | Zhang H.,Key Laboratory of Neural and Vascular Biology | Huang D.,Key Laboratory of Neural and Vascular Biology | Qi J.,Key Laboratory of Neural and Vascular Biology | And 4 more authors.
Pflugers Archiv European Journal of Physiology | Year: 2015

The Ca2+ activated Cl− channels (CaCCs) play a multitude of important physiological functions. A number of candidate proteins have been proposed to form CaCC, but only two families, the bestrophins and the TMEM16 proteins, recapitulate the properties of native CaCC in expression systems. Studies of endogenous CaCCs are hindered by the lack of specific pharmacology as most Cl− channel modulators lack selectivity and a systematic comparison of the effects of these modulators on TMEM16A and bestrophin is missing. In the present study, we studied seven Cl− channel inhibitors: niflumic acid (NFA), NPPB, flufenamic acid (FFA), DIDS, tannic acid, CaCCinh-A01 and T16Ainh-A01 for their effects on TMEM16A and bestrophin-1 (Best1) stably expressed in CHO (Chinese hamster ovary) cells using patch clamp technique. Among seven inhibitors studied, NFA showed highest selectivity for TMEM16A (IC50 of 7.40 ± 0.95 μM) over Best1 (IC50 of 102.19 ± 15.05 μM). In contrast, DIDS displayed a reverse selectivity inhibiting Best1 with IC50 of 3.93 ± 0.73 μM and TMEM16A with IC50 of 548.86 ± 25.57 μM. CaCCinh-A01 was the most efficacious blocker for both TMEM16A and Best1 channels. T16Ainh-A01 partially inhibited TMEM16A currents but had no effect on Best1 currents. Tannic acid, NPPB and FFA had variable intermediate effects. Potentiation of channel activity by some of these modulators and the effects on TMEM16A deactivation kinetics were also described. Characterization of Cl− channel modulators for their effects on TMEM16A and Best1 will facilitate future studies of native CaCCs. © 2014, Springer-Verlag Berlin Heidelberg.

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