Optogenetic manipulation of lysosomal physiology and autophagy-dependent clearance of amyloid beta.

Lysosomes are degradation centers of cells and intracellular hubs of signal transduction, nutrient sensing, and autophagy regulation. Dysfunction of lysosomes contributes to a variety of diseases, such as lysosomal storage diseases (LSDs) and neurodegeneration, but the mechanisms are not well understood. Altering lysosomal activity and examining its impact on the occurrence and development of disease is an important strategy for studying lysosome-related diseases. However, methods to dynamically regulate lysosomal function in living cells or animals are still lacking. Here, we constructed lysosome-localized optogenetic actuators, named lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2, to achieve optogenetic manipulation of lysosomes. These new actuators enable light-dependent control of lysosomal membrane potential, pH, hydrolase activity, degradation, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy through the mTOR pathway, promotes Aß clearance in an autophagy-dependent manner in cellular models, and alleviates Aß-induced paralysis in the Caenorhabditis elegans model of Alzheimer's disease. Our lysosomal optogenetic actuators supplement the optogenetic toolbox and provide a method to dynamically regulate lysosomal physiology and function in living cells and animals.

Metabolomic profiling of single enlarged lysosomes.

Lysosomes are critical for cellular metabolism and are heterogeneously involved in various cellular processes. The ability to measure lysosomal metabolic heterogeneity is essential for understanding their physiological roles. We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nanoESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. The accuracy and reliability of this technique were validated by supporting previous findings, such as the transportability of lysosomal cationic amino acids transporters such as PQLC2 and the lysosomal trapping of lysosomotropic, hydrophobic weak base drugs such as lidocaine. We derived metabolites from single lysosomes in various cell types and classified lysosomes into five major subpopulations based on their chemical and biological divergence. Senescence and carcinoma altered metabolic profiles of lysosomes in a type-specific manner. Thus, SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes.

Lysosomal cation channel TRPML1 suppression sensitizes acute myeloid leukemia cells to chemotherapeutics by inhibiting autophagy.

Despite the implementation of novel therapeutic regimens and extensive research efforts, chemoresistance remains a formidable challenge in the treatment of acute myeloid leukemia (AML). Notably, the involvement of lysosomes in chemoresistance has sparked interest in developing lysosome-targeted therapies to sensitize tumor cells to currently approved chemotherapy or as innovative pharmacological approaches. Moreover, as ion channels on the lysosomal membrane are critical regulators of lysosomal function, they present potential as novel targets for enhancing chemosensitivity. Here, we discovered that the expression of a lysosomal cation channel, namely transient receptor potential mucolipin 1 (TRPML1), was elevated in AML cells. Inhibiting TRPML1 individually does not impact the proliferation and apoptosis of AML cells. Importantly, inhibition of TRPML1 demonstrated the potential to modulate the sensitivity of AML cells to chemotherapeutic agents. Exploration of the underlying mechanisms revealed that suppression of TRPML1 impaired autophagy while concurrently increasing the production of reactive oxygen species (ROS) and ROS-mediated lipid peroxidation (Lipid-ROS) in AML cells. Finally, the knockdown of TRPML1 significantly reduced OCI-AML3 tumor growth following chemotherapy in a mouse model of human leukemia. In summary, targeting TRPML1 represents a promising approach for combination therapy aimed at enhancing chemosensitivity in treating AML.

Lysosomal K+ channel TMEM175 promotes apoptosis and aggravates symptoms of Parkinson's disease.

Lysosomes are degradative organelles and play vital roles in a variety of cellular processes. Ion channels on the lysosomal membrane are key regulators of lysosomal function. TMEM175 has been identified as a lysosomal potassium channel, but its modulation and physiological functions remain unclear. Here, we show that the apoptotic regulator Bcl-2 binds to and inhibits TMEM175 activity. Accordingly, Bcl-2 inhibitors activate the channel in a caspase-independent way. Increased TMEM175 function inhibits mitophagy, disrupts mitochondrial homeostasis, and increases production of reactive oxygen species (ROS). ROS further activates TMEM175 and thus forms a positive feedback loop to augment apoptosis. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD), knockout (KO) of TMEM175 mitigated motor impairment and dopaminergic (DA) neuron loss, suggesting that TMEM175-mediated apoptosis plays an important role in Parkinson's disease (PD). Overall, our study reveals that TMEM175 is an important regulatory site in the apoptotic signaling pathway and a potential therapeutic target for Parkinson's disease (PD).

A novel antisense long non-coding RNA participates in asexual and sexual reproduction by regulating the expression of GzmetE in Fusarium graminearum.

Fusarium graminearum is an important worldwide pathogen that causes Fusarium head blight in wheat, barley, maize and other grains. LncRNAs play important roles in many biological processes, but little is known about their functions and mechanisms in filamentous fungi. Here, we report that a natural antisense RNA, GzmetE-AS, is transcribed from the opposite strand of GzmetE. GzmetE encodes a homoserine O-acetyltransferase, which is important for sexual development and plant infection. The expression of GzmetE-AS was increased significantly during the conidiation stage, while GzmetE was upregulated in the late stage of sexual reproduction. Overexpression of GzmetE-AS inhibited the transcription of GzmetE. In contrast, the expression of GzmetE was significantly increased in GzmetE-AS transcription termination strain GzmetE-AS-T. Furthermore, GzmetE-AS-T produced more perithecia and facilitated the ascospore discharge, resembling the phenotype of GzmetE overexpressing strains. However, overexpression of GzmetE-AS in ∆dcl1/2 strain cannot inhibit the expression of GzmetE, and the GzmetE nat-siRNA is also significantly reduced in ∆dcl1/2 mutant. Taken together, we have identified a novel antisense lncRNA GzmetE-AS, which is involved in asexual and sexual reproduction by regulating its antisense gene GzmetE through RNAi pathway. Our findings reveal that the lncRNA plays critical roles in the development of F. graminearum.

TAB3 defect induces augmented cardioprotection loss from ischemic injury.

The transforming growth factor ß-activated kinase-binding protein 3 (TAB3) plays a crucial role in modulating cell apoptosis and proliferation in many diseases, including hepatocellular carcinoma, lung cancer, and intracerebral hemorrhage. However, the functional role of TAB3 in the heart is not well reported. In our study, we first investigated the role of TAB3 in ischemia heart diseases. For in vitro studies, cardiomyocytes (CMs) were isolated from both TAB3 knockout (KO) and wild-type (WT) mice, and the apoptosis ratios were tested after a 48-h ischemic stimulation. A proliferation test and tubeformation assay were underwent at normoxia condition. For in vivo studies, the MI model was completed for both TAB3 KO and WT mice. Echocardiography was evaluated at 3 days and 28 days post-MI, whereas the hemodynamics test was performed 28 days post-MI. The histology results of the apoptosis, proliferation of myocardium, neovasculation of microvessels, and infarct zone assessments were determined using terminal deoxynucleotidyl transferase dUTP nick end labeling staining, Ki67 immunostaining, α-SMA/CD31 immunostaining, and the Masson-Trichrome method, respectively. Expression changes of the related proteins caused by TAB3 deficiency were confirmed using both quantitative real-time polymerase chain reaction and immunoblotting. Our results indicate that the absence of TAB3 induced more CMs apoptosis, decrease cardiomyocyte proliferation, and weaker angiogenesis in vitro and in vivo. Worse cardiac function and enlarged scar formation were detected in TAB3 KO mice, and increased expression of active-caspase-3 and decreased expression of NF-κB/p65, Akt, Bcl-2/Bax, and VEGF occurred. In summary, our findings indicate that the absence of TAB3 plays a harmful role in ischemic heart disease.

Inhibition of lysosomal TRPML1 channel eliminates breast cancer stem cells by triggering ferroptosis.

Cancer stem cells (CSCs) are a sub-population of cells possessing high tumorigenic potential, which contribute to therapeutic resistance, metastasis and recurrence. Eradication of CSCs is widely recognized as a crucial factor in improving patient prognosis, yet the effective targeting of these cells remains a major challenge. Here, we show that the lysosomal cation channel TRPML1 represents a promising target for CSCs. TRPML1 is highly expressed in breast cancer cells and exhibits sensitivity to salinomycin, a drug known to selectively eliminate CSCs. Pharmacological inhibition and genetic depletion of TRPML1 promote ferroptosis in breast CSCs, reduce their stemness, and enhance the sensitivity of breast cancer cells to chemotherapy drug doxorubicin. The inhibition and knockout of TRPML1 also demonstrate significant suppression of tumor formation and growth in the mouse xenograft model. These findings suggest that targeting TRPML1 to eliminate CSCs may be an effective strategy for the treatment of breast cancer.

RNA-dependent RNA polymerases regulate ascospore discharge through the exonic-sRNA-mediated RNAi pathway.

Ascospores, forcibly released into the air from perithecia, are the primary inoculum for Fusarium head blight. In Fusarium graminearum, the biological functions of four RNA-dependent RNA polymerases (RdRPs) (Fgrdrp1-4) have been reported, but their regulatory mechanisms are poorly understood and the function of Fgrdrp5 is still unknown. In this study, we found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays an important role in ascospore discharge, and they all participate in the generation of turgor pressure in a polyol-dependent manner. Moreover, these three genes all affect the maturation of ascospores. Deep sequencing and co-analysis of small RNA and mRNA certified that Fgrdrp1, Fgrdrp2, and Fgrdrp5 partly share their functions in the biogenesis and accumulation of exonic small interference RNA (ex-siRNA), and these three RdRPs negatively regulate the expression levels of ex-siRNA corresponding genes, including certain genes associated with ascospore development or discharge. Furthermore, the differentially expressed genes of deletion mutants, those involved in lipid and sugar metabolism or transport as well as sexual development-related transcription factors, may also contribute to the defects in ascospore maturation or ascospore discharge. In conclusion, our study suggested that the components of the dicer-dependent ex-siRNA-mediated RNA interference pathway include at least Fgrdrp1, Fgrdrp2, and Fgrdrp5. IMPORTANCE: We found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays important roles in ascospore maturation and ascospore discharge of Fusarium graminearum. These three RNA-dependent RNA polymerases participate in the biogenesis and accumulation of exonic small interference RNA and then regulate ascospore discharge.

Exploring the effects of physical exercise on inferiority feeling in children and adolescents with disabilities: a test of chain mediated effects of self-depletion and self-efficacy.

Objective: The purpose of this study was to investigate the effects of physical exercise on inferiority feeling of children and adolescents with disabilities and its mechanism of action, as well as the mediating role of self depletion and self-efficacy. Methods: The following scales were administered to 546 children and adolescents with disabilities (mean age 15.6 years): The Feelings of Inadequacy Scale, (FIS), the Self-Regulation Fatigue Scale (S-RFS), the General Self-Efficacy Scale (GSES), and the Physical Exercise Rating Scale. Results: (1) Physical exercise can directly and negatively predict inferiority feeling, self-depletion, and can directly and positively predict self-efficacy; self-depletion can directly and negatively predict self-efficacy. Similarly, self-depletion positively predicts inferiority feeling; physical exercise and self-efficacy can also directly and negatively predict inferiority feeling. (2) The indirect effect of the path with self-depletion as the mediating variable was - 0.05, the indirect effect of the path with self-efficacy as the mediating variable was - 0.09, and the indirect effect of the path with self-depletion and self-efficacy as the mediating variables was - 0.04. (3) The sum of all indirect effects was - 0.18, and the three indirect effects accounted for 15.6%, 28.1%, and 12.5% of the total effect, with mediating effect was 56.2%. Conclusion: Physical exercise can indirectly predict inferiority feeling in children and adolescents with disabilities through the independent mediation of self-depletion and self-efficacy, as well as through the chain mediation of both. This study supports that moderate physical exercise has a positive effect on the mental health of children and adolescents with disabilities, and that reducing self-depletion and improving self-efficacy are important ways to prevent inferiority feeling among children and adolescents with disabilities. It reveals the relationship between physical exercise and inferiority feeling and its mechanism of action, and further improves the research on the effect of physical exercise on inferiority feeling of children and adolescents with disabilities.

lncRsp1, a long noncoding RNA, influences Fgsp1 expression and sexual reproduction in Fusarium graminearum.

Long noncoding RNAs (lncRNAs) are crucial regulators of gene expression in many biological processes, but their biological functions remain largely unknown, especially in fungi. Fusarium graminearum is an important pathogen that causes the destructive disease Fusarium head blight (FHB) or head scab disease on wheat and barley. In our previous RNA sequencing (RNA-Seq) study, we discovered that lncRsp1 is an lncRNA that is located +99 bp upstream of a putative sugar transporter gene, Fgsp1, with the same transcription direction. Functional studies revealed that ΔlncRsp1 and ΔFgsp1 were normal in growth and conidiation but had defects in ascospore discharge and virulence on wheat coleoptiles. Moreover, lncRsp1 and Fgsp1 were shown to negatively regulate the expression of several deoxynivalenol (DON) biosynthesis genes, TRI4, TRI5, TRI6, and TRI13, as well as DON production. Further analysis showed that the overexpression of lncRsp1 enhanced the ability of ascospore release and increased the mRNA expression level of the Fgsp1 gene, while lncRsp1-silenced strains reduced ascospore discharge and inhibited Fgsp1 expression during the sexual reproduction stage. In addition, the lncRsp1 complementary strains lncRsp1-LC-1 and lncRsp1-LC-2 restored ascospore discharge to the level of the wild-type strain PH-1. Taken together, our results reveal the distinct and specific functions of lncRsp1 and Fgsp1 in F. graminearum and principally demonstrate that lncRsp1 can affect the release of ascospores by regulating the expression of Fgsp1.

[Effects of metoprolol on electrophysiology of ischemic and anoxic myocardium in diabetic rats].

OBJECTIVE: To investigate the effects of metoprolol on electrophysiology of ischemic and anoxic myocardium in diabetic rats. METHODS: Forty Sprague-Dawley (SD) rats were divided into 4 groups: diabetes group; diabetes and ablation of left sympathetic nerve group; diabetes and metoprolol group and sham group. The diabetes model was induced by intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). The ventricular diastolic effective threshold (DET), effective refractive period (ERP), and Ventricular fibrillation threshold (VFT) were measured. The serum concentration of nerve growth factor (NGF) was measured. RESULTS: Metoprolol increased DET of ischemic and anoxic myocardium in diabetic rats. The ablation of the left sympathetic nerve increased VFT of diabetic rats. VFT in metoprolo group was significantly increased compared to diabetes group after ischemia. The concentrations of NGF in diabetic group and metoprolol group were higher than those in sham group. There were no difference in NGF levels between ablation of left sympathetic nerve group and sham group. CONCLUSION: The remodeling of sympathetic nerve affects the electrophysiology of ischemic myocardium of diabetic rats. Metoprolol can increase the VFT and decrease the excitation threshold of the ischemic myocardium in diabetic rats.

CLN7 is an organellar chloride channel regulating lysosomal function.

Neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive lysosomal storage diseases. One variant form of late-infantile NCL (vLINCL) is caused by mutations of a lysosomal membrane protein CLN7, the function of which has remained unknown. Here, we identified CLN7 as a novel endolysosomal chloride channel. Overexpression of CLN7 increases endolysosomal chloride currents and enlarges endolysosomes through a Ca2+/calmodulin-dependent way. Human CLN7 and its yeast homolog exhibit characteristics of chloride channels and are sensitive to chloride channel blockers. Moreover, CLN7 regulates lysosomal chloride conductance, luminal pH, and lysosomal membrane potential and promotes the release of lysosomal Ca2+ through transient receptor potential mucolipin 1 (TRPML1). Knocking out CLN7 causes pathological features that are similar to those of patients with vLINCL, including retinal degeneration and autofluorescent lipofuscin. The pathogenic mutations in CLN7 lead to a decrease in chloride permeability, suggesting that reconstitution of lysosomal Cl− homeostasis may be an effective strategy for the treatment of vLINCL.

Hydrocarbon Dew Point Measurement and Model Evaluation of Synthetic and Real Natural Gases.

Hydrocarbon dew point (HCDP) is one of the most important quality specifications of natural gas. Measuring and predicting the HCDP accurately are essential for the natural gas industry. However, the comprehensive experimental HCDP curve data are still rare, and knowledge about adopting proper prediction models remains unclear. In view of this, HCDP determination work by use of an improved test system and model evaluation based on more than 1000 dew points data have been done to improve the aforementioned dilemma. HCDP curve data of three gravimetrically prepared synthetic natural gases (SNGs) and one real gas (RG) are determined first. Then, one set of data containing 712 dew points from 28 SNGs and 334 dew points from 14 RGs is used to evaluate the performance of eight different HCDP prediction models including Soave-Redlich-Kwong (SRK), SRK-Twu, Peng-Robinson (PR), Twu-Sim-Tassone (TST), predictive SRK (PSRK), GERG-2008, PSRK, and perturbed-chain statistical associating fluid theory (PC-SAFT) models. Considerable prediction deviation of these models in the high-pressure region (pressure above 6.0 MPa) is observed compared to that in the low-pressure region (under 6.0 MPa), and the reasons for that difference are discussed. Evaluation results reveal that among the eight prediction models, GERG-2008 has the best performance (overall average absolute deviation (AAD): 1.44 °C) for SNGs, and PSRK and SRK-Twu fits the experimental data best for RGs (overall AAD: 2.50 °C). Therefore, GERG-2008 is recommended for HCDP prediction of relatively lean gas, whereas PSRK and SRK-Twu are recommended for calculating the HCDP of relatively heavy natural gases in low-pressure and high-pressure regions, respectively.

A conserved arginine/lysine-based motif promotes ER export of KCNE1 and KCNE2 to regulate KCNQ1 channel activity.

KCNE ß-subunits play critical roles in modulating cardiac voltage-gated potassium channels. Among them, KCNE1 associates with KCNQ1 channel to confer a slow-activated IKs current, while KCNE2 functions as a dominant negative modulator to suppress the current amplitude of KCNQ1. Any anomaly in these channels will lead to serious myocardial diseases, such as the long QT syndrome (LQTS). Trafficking defects of KCNE1 have been reported to account for the pathogenesis of LQT5. However, the molecular mechanisms underlying KCNE forward trafficking remain elusive. Here, we describe an arginine/lysine-based motif ([R/K](S)[R/K][R/K]) in the proximal C-terminus regulating the endoplasmic reticulum (ER) export of KCNE1 and KCNE2 in HEK293 cells. Notably, this motif is highly conserved in the KCNE family. Our results indicate that the forward trafficking of KCNE2 controlled by the motif (KSKR) is essential for suppressing the cell surface expression and current amplitude of KCNQ1. Unlike KCNE2, the motif (RSKK) in KCNE1 plays important roles in modulating the gating of KCNQ1 in addition to mediating the ER export of KCNE1. Furthermore, truncations of the C-terminus did not reduce the apparent affinity of KCNE2 for KCNQ1, demonstrating that the rigid C-terminus of KCNE2 may not physically interact with KCNQ1. In contrast, the KCNE1 C-terminus is critical for its interaction with KCNQ1. These results contribute to the understanding of the mechanisms of KCNE1 and KCNE2 membrane targeting and how they coassemble with KCNQ1 to regulate the channels activity.

Dicer-Like Proteins Regulate Sexual Development via the Biogenesis of Perithecium-Specific MicroRNAs in a Plant Pathogenic Fungus Fusarium graminearum.

Ascospores act as the primary inoculum of Fusarium graminearum, which causes the destructive disease Fusarium head blight (FHB), or scab. MicroRNAs (miRNAs) have been reported in the F. graminearum vegetative stage, and Fgdcl2 is involved in microRNA-like RNA (milRNA) biogenesis but has no major impact on vegetative growth, abiotic stress or pathogenesis. In the present study, we found that ascospore discharge was decreased in the Fgdcl1 deletion mutant, and completely blocked in the double-deletion mutant of Fgdcl1 and Fgdcl2. Besides, more immature asci were observed in the double-deletion mutant. Interestingly, the up-regulated differentially expressed genes (DEGs) common to ΔFgdcl1 and ΔFgdcl1/2 were related to ion transmembrane transporter and membrane components. The combination of small RNA and transcriptome sequencing with bioinformatics analysis predicted 143 novel milRNAs in wild-type perithecia, and 138 of these milRNAs partly or absolutely depended on Fgdcl1, while only 5 novel milRNAs were still obtained in the Fgdcl1 and Fgdcl2 double-deletion mutant. Furthermore, 117 potential target genes were predicted. Overall, Fgdcl1 and Fgdcl2 genes were partly functionally redundant in ascospore discharge and perithecium-specific milRNA generation in F. graminearum, and these perithecium-specific milRNAs play potential roles in sexual development.

Association of the Endothelial Nitric Oxide Synthase Gene T786C Polymorphism with In-Stent Restenosis in Chinese Han Patients with Coronary Artery Disease Treated with Drug-Eluting Stent.

BACKGROUND AND AIM: Many studies have reported that genetic variants correlate with higher risk for coronary artery disease (CAD) or in-stent restenosis (ISR) after bare metal stent (BMS) implantation. However, there is limited data assessing the impact of these variants on ISR in patients treated with drug-eluting stent (DES). The purpose of this study was to investigate the effects of genetic risk factors on ISR in Chinese Han patients treated with DES. METHODS: A total of 425 patients with a diagnosis of CAD who underwent successful revascularization in native coronary arteries with DES were included in this retrospective study. Genotyping was performed on six single nucleotide polymorphisms (SNPs) in the endothelial nitric oxide synthase gene (eNOS), the angiotensin converting enzyme gene (ACE), the angiotensin II type 1 receptor gene (AT1R), the transforming growth factor beta gene (TGF-ß), and the vascular endothelial growth factor gene (VEGF). Quantitative coronary angiography (QCA) was performed during the follow-up period to detect ISR. Logistic regression models were used to test for association. RESULTS: Fifty-four patients (12.7%) developed ISR during the follow-up period. Of the six analyzed SNPs, the frequency of the C allele of T786C polymorphism in eNOS was significantly higher in the ISR group (22.2%) compared to the non-ISR group (12.7%) (p<0.01). In the ISR group, the frequency of the TT, TC, and CC genotypes was 61.1%, 33.3%, and 5.6%, respectively, and in the non-ISR group, the frequencies were 76.8%, 21.0%, and 2.2%, respectively. The multivariable analysis adjusted for potential confounders and revealed that the T786C polymorphism increased the risk of ISR in both additive and dominant models with odds ratios of 1.870 (95% confidence interval [CI]: 1.079-3.240, p = 0.03) and 2.045 (95% CI: 1.056-3.958, p = 0.03), respectively. CONCLUSION: The eNOS T786C polymorphism was associated with ISR in Chinese Han patients treated with DES. Genotyping may be helpful to identify patients with higher risks of ISR after DES implantation.

Extrapolating microdomain Ca(2+) dynamics using BK channels as a Ca(2+) sensor.

Ca(2+) ions play crucial roles in mediating physiological and pathophysiological processes, yet Ca(2+) dynamics local to the Ca(2+) source, either from influx via calcium permeable ion channels on plasmic membrane or release from internal Ca(2+) stores, is difficult to delineate. Large-conductance calcium-activated K(+) (BK-type) channels, abundantly distribute in excitable cells and often localize to the proximity of voltage-gated Ca(2+) channels (VGCCs), spatially enabling the coupling of the intracellular Ca(2+) signal to the channel gating to regulate membrane excitability and spike firing patterns. Here we utilized the sensitivity and dynamic range of BK to explore non-uniform Ca(2+) local transients in the microdomain of VGCCs. Accordingly, we applied flash photolysis of caged Ca(2+) to activate BK channels and determine their intrinsic sensitivity to Ca(2+). We found that uncaging Ca(2+) activated biphasic BK currents with fast and slow components (time constants being τf ≈ 0.2 ms and τs ≈ 10 ms), which can be accounted for by biphasic Ca(2+) transients following light photolysis. We estimated the Ca(2+)-binding rate constant kb (≈1.8 × 10(8) M(-1) s(-1)) for mSlo1 and further developed a model in which BK channels act as a calcium sensor capable of quantitatively predicting local microdomain Ca(2+) transients in the vicinity of VGCCs during action potentials.

Single Channel Recordings Reveal Differential ß2 Subunit Modulations Between Mammalian and Drosophila BKCa(ß2) Channels.

Large-conductance Ca2+- and voltage-activated potassium (BK) channels are widely expressed in tissues. As a voltage and calcium sensor, BK channels play significant roles in regulating the action potential frequency, neurotransmitter release, and smooth muscle contraction. After associating with the auxiliary ß2 subunit, mammalian BK(ß2) channels (mouse or human Slo1/ß2) exhibit enhanced activation and complete inactivation. However, how the ß2 subunit modulates the Drosophila Slo1 channel remains elusive. In this study, by comparing the different functional effects on heterogeneous BK(ß2) channel, we found that Drosophila Slo1/ß2 channel exhibits "paralyzed"-like and incomplete inactivation as well as slow activation. Further, we determined three different modulations between mammalian and Drosophila BK(ß2) channels: 1) dSlo1/ß2 doesn't have complete inactivation. 2) ß2(K33,R34,K35) delays the dSlo1/Δ3-ß2 channel activation. 3) dSlo1/ß2 channel has enhanced pre-inactivation than mSlo1/ß2 channel. The results in our study provide insights into the different modulations of ß2 subunit between mammalian and Drosophila Slo1/ß2 channels and structural basis underlie the activation and pre-inactivation of other BK(ß) complexes.

Differential modulations of KCNQ1 by auxiliary proteins KCNE1 and KCNE2.

KCNQ1 channels play vital roles in cardiovascular, gastric and other systems. The conductance and dynamics of KCNQ1 could be modulated by different single transmembrane helical auxiliary proteins (such as KCNE1, KCNE2 and others). In this study, detail KCNQ1 function modulations by different regions of KCNE1 or KCNE2 were examined using combinational methods of electrophysiology, immunofluorescence, solution NMR and related backbone flexibility analysis. In the presence of KCNE2 N-terminus, decreased surface expression and consequent low activities of KCNQ1 were observed. The transmembrane domains (TMDs) of KCNE1 and KCNE2 were illustrated to associate with the KCNQ1 channel in different modes: Ile64 in KCNE2-TMD interacting with Phe340 and Phe275 in KCNQ1, while two pairs of interacting residues (Phe340-Thr58 and Ala244-Tyr65) in the KCNQ1/KCNE1 complex. The KCNE1 C-terminus could modulate gating property of KCNQ1, whereas KCNE2 C-terminus had only minimal influences on KCNQ1. All of the results demonstrated different KCNQ1 function modulations by different regions of the two auxiliary proteins.

Inter-α/ß subunits coupling mediating pre-inactivation and augmented activation of BKCa(ß2).

Large-conductance calcium-activated potassium (BK) channels regulate the electric properties and neurotransmitter release in excitable cells. Its auxiliary ß2 subunits not only enhance gating, but also confer inactivation via a short-lived preinactivated state. However, the mechanism of enhancement and preinactivation of BK channels by ß2 remains elusive. Using our newly developed methods, we demonstrated that electrostatic forces played a crucial role in forming multiple complementary pairs of binding sites between α and ß subunits including a "PI site" required for channel preinactivation, an "E site" enhancing calcium sensitivity and an "ECaB" coupling site transferring force to gate from the Ca(2+)-bowl via the ß2(K33, R34, K35), E site and S6-C linker, independent of another Ca(2+) binding site mSlo1(D362,D367). A comprehensive structural model of the BK(ß2) complex was reconstructed based on these functional studies, which paves the way for a clearer understanding of the structural mechanisms of activation and preinactivation of other BK(ß) complexes.