Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

Many efforts have been made to understand excitotoxicity and develop neuroprotectants for the therapy of ischemic stroke. The narrow treatment time window is still to be solved. Given that the ischemic core expanded over days, treatment with an extended time window is anticipated. Bestrophin 1 (BEST1) belongs to a bestrophin family of calcium-activated chloride channels. We revealed an increase in neuronal BEST1 expression and function within the peri-infarct from 8 to 48 h after ischemic stroke in mice. Interfering the protein expression or inhibiting the channel function of BEST1 by genetic manipulation displayed neuroprotective effects and improved motor functional deficits. Using electrophysiological recordings, we demonstrated that extrasynaptic glutamate release through BEST1 channel resulted in delayed excitotoxicity. Finally, we confirmed the therapeutic efficacy of pharmacological inhibition of BEST1 during 6-72 h post-ischemia in rodents. This delayed treatment prevented the expansion of infarct volume and the exacerbation of neurological functions. Our study identifies the glutamate-releasing BEST1 channel as a potential therapeutic target against ischemic stroke with a wide time window.

Establishment of a cell model for high-throughput TRPV4 regulator screening based on CaCC / 解放军医学杂志

Objective To construct a high-throughput screening model for transient receptor potential vanilloid 4 (TRPV4) channel modulators based on calcium-activated chloride channels (CaCC). Methods RT-PCR was used to detect the endogenous expression of TRPV4 in Fischer rat thyroid (FRT) cells. The PCR products obtained were subjected to nucleic acid sequencing using gel-recovery technology. Western blotting was employed to detect the expression of TRPV4 protein in FRT cells. The liposome transfection method was applied to construct the FRT cell model that co-expressed anoctamin 1 (ANO1) and YFP-H148Q/ I152L. The expressions of ANO1 and YFP-H148Q/I152L in cells were identified by the inverted fluorescence microscope and the fluorescence quenching kinetics test. After adding TRPV4 activators and inhibitors, the fluorescence quenching kinetics experiment was used to test whether the model could screen TRPV4 modulators. The Fura-2 fluorescent probe method was applied to detect the calcium concentration in cells after adding TRPV4 activators; The Z' factor was calculated to evaluate the sensitivity and specificity of the cell model. Results RT-PCR and Western blotting confirmed the endogenous expression of TRPV4 in FRT cells; ANO1 was clearly expressed on the FRT cell membrane and YFP-H148Q/I152L was clearly expressed in the cytoplasm of FRT cells under the inverted fluorescence microscope. The FRT cell model co-expressing ANO1 and YFP-H148Q/I152L was successfully constructed. Fluorescence quenching kinetics experiments confirmed that the model could screen TRPV4 regulators, and the slope value of fluorescence change and the concentration of TRPV4 regulator concentration were in a dose-dependent manner. The model could sensitively detect changes in intracellular calcium concentration, and the slope value could reflect intracellular calcium concentration. The Z' factor was 0.728, which demonstrates its capacity for high-throughput screening. Conclusions We successfully constructed a high-throughput model that could screen TRPV4 modulators sensitively and efficiently.

Calcium Signaling in Salivary Secretion

Calcium has versatile roles in diverse physiological functions. Among these functions, intracellular Ca²⁺ plays a key role during the secretion of salivary glands. In this review, we introduce the diverse cellular components involved in the saliva secretion and related dynamic intracellular Ca²⁺ signals. Calcium acts as a critical second messenger for channel activation, protein translocation, and volume regulation, which are essential events for achieving the salivary secretion. In the secretory process, Ca²⁺ activates K⁺ and Cl⁻ channels to transport water and electrolyte constituting whole saliva. We also focus on the Ca²⁺ signals from intracellular stores with discussion about detailed molecular mechanism underlying the generation of characteristic Ca²⁺ patterns. In particular, inositol triphosphate signal is a main trigger for inducing Ca²⁺ signals required for the salivary gland functions. The biphasic response of inositol triphosphate receptor and Ca²⁺ pumps generate a self-limiting pattern of Ca²⁺ efflux, resulting in Ca²⁺ oscillations. The regenerative Ca²⁺ oscillations have been detected in salivary gland cells, but the exact mechanism and function of the signals need to be elucidated. In future, we expect that further investigations will be performed toward better understanding of the spatiotemporal role of Ca²⁺ signals in regulating salivary secretion.

Expression and identification of ANO1 in mouse cardiomyocytes / 中国病理生理杂志

AIM:To explore the expression of anoctamin 1 (ANO1), one of calcium-activated chloride chan-nels ( CaCCs) , in mouse cardiomyocytes and its functional properties.METHODS:The cardiomyocytes from the myocar-dial tissues of C57BL/6 mice were isolated with enzyme and purified by the differential adherent method.The cells were stained with monoclonal anti-sarcomeric actin and Cy3 to evaluate the purity of the myocardial cells.RT-PCR was used to detect the mRNA expression of ANO1 in the mouse cardiomyocytes.The protein expression of ANO1 in the mouse cardio-myocytes was determined by Western blotting analysis.The fluorescence quenching kinetics experiment was used to identify the ion transport properties of ANO1 in the mouse cardiomyocytes.RESULTS: The results of RT-PCR confirmed that ANO1 was expressed in freshly isolated myocardial cells.The results of Western blotting clearly demonstrated the protein expression of ANO1 in primarily cultured myocardial cells.Fluorescence quenching kinetics experiment on freshly isolated single myocardial cell revealed a pronounced outward rectifying property of the ANO1.The functional properties were simi-lar to the classic CaCCs.CONCLUSION:ANO1 expression was identified in the mouse myocardial cells.The function of CaCCs was generated by ANO1, suggesting that ANO1 is the molecular basis of CaCCs.

Research progress of transmembrane protein 16a in cardiovascular system / 中华实用儿科临床杂志

Calcium-activated chloride channels(CaCCs) play an important role in cardiovascular system,including participating in a variety of physiological functions and being associated with the pathology progress of many cardiovascular diseases.With the development of research,transmembrane protein 16a (TMEM16A) has recently been identified as the molecular basis of CaCCs.Since the study of TMEM16A has got some achievement,especially in the section of cardiovascular system,its main research progress and key points from previous researches are summarized,in which the expression and physiological function of TMEM16A,as well as its clinical pathological correlations with some cardiovascular diseases are told about.Besides,the research prospect of TMEM16A in targeting therapy of cardiovascular disease is also discussed.

Expression of TMEM16 A as a calcium-activated chloride channel in Fis-cher rat thyroid follicular epithelial cells and its electrophysiologic pro-perties / 中国病理生理杂志

AIM:To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thy-roid follicular epithelial ( FRT) cells and its electrophysiologic properties .METHODS: The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection .In order to ob-tain the high efficiency of gene transfection and expression , the quantity and ratio of lipid/DNA complexes were optimized . The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence .The expression and location of TMEM 16A in the FRT cells were observed under an in-verted fluorescence microscope .TMEM16A protein was associated with calcium-dependent chloride current , as measured with halide-sensitive fluorescent protein and patch-clamp technique .RESULTS: The results of double digestion and se-quencing indicated that TMEM16A was cloned into pUB6/V5.The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A.The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM 16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L.CONCLUSION:The protein expression of TMEM16A in the FRT cells was observed.TMEM16A is the molecular identity of calcium-activated chloride channels .

TMEM16 proteins: the long awaited calcium-activated chloride channels?

Currents mediated by calcium-activated chloride channels (CaCCs), observed for the first time in Xenopus oocytes, have been recorded in many cells and tissues ranging from different types of neurons to epithelial and muscle cells. CaCCs play a role in the regulation of excitability in neurons including sensory receptors. In addition, they are crucial mediators of chloride movements in epithelial cells where their activity regulates electrolyte and fluid transport. The roles of CaCCs, particularly in epithelia, are briefly reviewed with emphasis on their function in secretory epithelia. The recent identification by three independent groups, using different strategies, of TMEM16A as the molecular counterpart of the CaCC is discussed. TMEM16A is part of a family that has 10 other members in mice. The discovery of the potential TMEM16 anion channel activity opens the way for the molecular investigation of the role of these anion channels in specific cells and in organ physiology and pathophysiology. The identification of TMEM16A protein as a CaCC chloride channel molecule represents a great triumph of scientific perseverance and ingenuity. The varied approaches used by the three independent research groups also augur well for the solidity of the discovery.

Different mRNA expressions of calcium activated channels in normal and instable detrusors / 第三军医大学学报

Objective To study the different mRNA expressions of big and small conductance calcium activated potassium channels (Bkca and Skca), and calcium activated chloride channels (Clca) in normal and instable detrusors. Methods Model of bladder outlet obstruction (BOO) of female Wistar rats was prepared by ligating the proximal urethra in the perineum. Detrusor instability (DI) was confirmed by conscious cystometry. The mRNA was extracted from the detrusors of normal and DI rats for the detection of the expressions of Bkca, Skca2, Skca3, and Clca by RT PCR. The different channel expression between normal and instable detrusors was identified by gel imaging. Results The incidence of DI in BOO rats was 76.17%. Bladder capacity and the maximal detrusor pressure increased significantly ( P