Negative Charges in the Transmembrane Domains of the HERG K Channel Are Involved in the Activation- and Deactivation-gating Processes

The transmembrane domains of HERG (S1–S3) contain six negative charges: three are conserved in all voltage-gated K channels (D456 and D466 in S2, D501 in S3) and three are unique to the EAG family (D411 in S1, D460 in S2, and D509 in S3). We infer the functional role of these aspartates by studying how substituting them with cysteine, one at a time, affects the channel function. D456C is not functional, suggesting that this negative charge may play a critical role in channel protein folding during biogenesis, as has been shown for its counterpart in the Shaker channel. Data from the other five functional mutants suggest that D411 can stabilize the HERG channel in the closed state, while D460 and D509 have the opposite effect. D466 and D501 both may contribute to voltage-sensing during the activation process. On the other hand, all five aspartates work in a concerted fashion in contributing to the slow deactivation process of the HERG channel. Accessibility tests of the introduced thiol groups to extracellular MTS reagents indicate that water-filled crevices penetrate deep into the HERG protein core, reaching the cytoplasmic halves of S1 and S2. At these deep locations, accessibility of 411C and 466C to the extracellular aqueous phase is voltage dependent, suggesting that conformational changes occur in S1 and S2 or the surrounding crevices during gating. Increasing extracellular [H+] accelerates HERG deactivation. This effect is suppressed by substituting the aspartates with cysteine, suggesting that protonation of these aspartates may contribute to the signaling pathway whereby external [H+] influences conformational changes in the channel's cytoplasmic domains (where deactivation takes place). There is no evidence for a metal ion binding site coordinated by negative charges in the transmembrane domains of HERG, as the one described for the EAG channel

Structural and Functional Role of the Extracellular S5-P Linker in the HERG Potassium Channel

C-type inactivation in the HERG channel is unique among voltage-gated K channels in having extremely fast kinetics and strong voltage sensitivity. This suggests that HERG may have a unique outer mouth structure (where conformational changes underlie C-type inactivation), and/or a unique communication between the outer mouth and the voltage sensor. We use cysteine-scanning mutagenesis and thiol-modifying reagents to probe the structural and functional role of the S5-P (residues 571–613) and P-S6 (residues 631–638) linkers of HERG that line the outer vestibule of the channel. Disulfide formation involving introduced cysteine side chains or modification of side chain properties at “high-impact” positions produces a common mutant phenotype: disruption of C-type inactivation, reduction of K+ selectivity, and hyperpolarizing shift in the voltage-dependence of activation. In particular, we identify 15 consecutive positions in the middle of the S5-P linker (583–597) where side chain modification has marked impact on channel function. Analysis of the degrees of mutation-induced perturbation in channel function along 583–597 reveals an α-helical periodicity. Furthermore, the effects of MTS modification suggest that the NH2-terminal of this segment (position 584) may be very close to the pore entrance. We propose a structural model for the outer vestibule of the HERG channel, in which the 583–597 segment forms an α-helix. With the NH2 terminus of this helix sitting at the edge of the pore entrance, the length of the helix (∼20 Å) allows its other end to reach and interact with the voltage-sensing domain. Therefore, the “583–597 helix” in the S5-P linker of the HERG channel serves as a bridge of communication between the outer mouth and the voltage sensor, that may make important contribution to the unique C-type inactivation phenotype

KCNQ1 and KCNE1 in the IKs Channel Complex Make State-dependent Contacts in their Extracellular Domains

KCNQ1 and KCNE1 (Q1 and E1) associate to form the slow delayed rectifier IKs channels in the heart. A short stretch of eight amino acids at the extracellular end of S1 in Q1 (positions 140–147) harbors six arrhythmia-associated mutations. Some of these mutations affect the Q1 channel function only when coexpressed with E1, suggesting that this Q1 region may engage in the interaction with E1 critical for the IKs channel function. Identifying the Q1/E1 contact points here may provide new insights into how the IKs channel operates. We focus on Q1 position 145 and E1 positions 40–43. Replacing all native cysteine (Cys) in Q1 and introducing Cys into the above Q1 and E1 positions do not significantly perturb the Q1 channel function or Q1/E1 interactions. Immunoblot experiments on COS-7 cells reveal that Q1 145C can form disulfide bonds with E1 40C and 41C, but not E1 42C or 43C. Correspondingly, voltage clamp experiments in oocytes reveal that Q1 145C coexpressed with E1 40C or E1 41C manifests unique gating behavior and DTT sensitivity. Our data suggest that E1 40C and 41C come close to Q1 145C in the activated and resting states, respectively, to allow disulfide bond formation. These data and those in the literature lead us to propose a structural model for the Q1/E1 channel complex, in which E1 is located between S1, S4, and S6 of three separate Q1 subunits. We propose that E1 is not a passive partner of the Q1 channel, but instead can engage in molecular motions during IKs gating

Building KCNQ1/KCNE1 Channel Models and Probing their Interactions by Molecular-Dynamics Simulations

The slow delayed rectifier (IKs) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of IKs-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in IKs channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance

Long-Term Fish Oil Supplementation Induces Cardiac Electrical Remodeling by Changing Channel Protein Expression in the Rabbit Model

Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks) affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s)/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography - mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (ICaL) and a decrease in the transient outward current (Ito) in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in ICaL and Ito were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1). We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes

TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes.

A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea

Overexpression of proto-oncogene FBI-1 activates membrane type 1-matrix metalloproteinase in association with adverse outcome in ovarian cancers

<p>Abstract</p> <p>Background</p> <p>FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) is a member of the POK (POZ and Kruppel) family of transcription factors and play important roles in cellular differentiation and oncogenesis. Recent evidence suggests that FBI-1 is expressed at high levels in a subset of human lymphomas and some epithelial solid tumors. However, the function of FBI-1 in human ovarian cancers remains elusive.</p> <p>Results</p> <p>In this study, we investigated the role of FBI-1 in human ovarian cancers, in particularly, its function in cancer cell invasion via modulating membrane type 1-matrix metalloproteinase (MT1-MMP). Significantly higher FBI-1 protein and mRNA expression levels were demonstrated in ovarian cancers samples and cell lines compared with borderline tumors and benign cystadenomas. Increased FBI-1 mRNA expression was correlated significantly with gene amplification (P = 0.037). Moreover, higher FBI-1 expression was found in metastatic foci (P = 0.036) and malignant ascites (P = 0.021), and was significantly associated with advanced stage (P = 0.012), shorter overall survival (P = 0.032) and disease-free survival (P = 0.016). <it>In vitro</it>, overexpressed FBI-1 significantly enhanced cell migration and invasion both in OVCA 420 and SKOV-3 ovarian carcinoma cells, irrespective of <it>p53 </it>status, accompanied with elevated expression of MT1-MMP, but not MMP-2 or TIMP-2. Moreover, knockdown of MT1-MMP abolished FBI-1-mediated cell migration and invasion. Conversely, stable knockdown of FBI-1 remarkably reduced the motility of these cells with decreased expression of MT1-MMP. Promoter assay and chromatin immunoprecipitation study indicated that FBI-1 could directly interact with the promoter spanning ~600bp of the 5'-flanking sequence of MT1-MMP and enhanced its expression in a dose-dependent manner. Furthermore, stable knockdown and ectopic expression of FBI-1 decreased and increased cell proliferation respectively in OVCA 420, but not in the p53 null SKOV-3 cells.</p> <p>Conclusions</p> <p>Our results suggested an important role of FBI-1 in ovarian cancer cell proliferation, cell mobility, and invasiveness, and that FBI-1 can be a potential target of chemotherapy.</p

Large-scale plasma proteomic profiling identifies a high-performance biomarker panel for Alzheimer's disease screening and staging

INTRODUCTION: Blood proteins are emerging as candidate biomarkers for Alzheimer's disease (AD). We systematically profiled the plasma proteome to identify novel AD blood biomarkers and develop a high-performance, blood-based test for AD. METHODS: We quantified 1160 plasma proteins in a Hong Kong Chinese cohort by high-throughput proximity extension assay and validated the results in an independent cohort. In subgroup analyses, plasma biomarkers for amyloid, tau, phosphorylated tau, and neurodegeneration were used as endophenotypes of AD. RESULTS: We identified 429 proteins that were dysregulated in AD plasma. We selected 19 “hub proteins” representative of the AD plasma protein profile, which formed the basis of a scoring system that accurately classified clinical AD (area under the curve = 0.9690–0.9816) and associated endophenotypes. Moreover, specific hub proteins exhibit disease stage-dependent dysregulation, which can delineate AD stages. DISCUSSION: This study comprehensively profiled the AD plasma proteome and serves as a foundation for a high-performance, blood-based test for clinical AD screening and staging

Towards a large-scale quantum simulator on diamond surface at room temperature

Strongly-correlated quantum many-body systems exhibits a variety of exotic phases with long-range quantum correlations, such as spin liquids and supersolids. Despite the rapid increase in computational power of modern computers, the numerical simulation of these complex systems becomes intractable even for a few dozens of particles. Feynman's idea of quantum simulators offers an innovative way to bypass this computational barrier. However, the proposed realizations of such devices either require very low temperatures (ultracold gases in optical lattices, trapped ions, superconducting devices) and considerable technological effort, or are extremely hard to scale in practice (NMR, linear optics). In this work, we propose a new architecture for a scalable quantum simulator that can operate at room temperature. It consists of strongly-interacting nuclear spins attached to the diamond surface by its direct chemical treatment, or by means of a functionalized graphene sheet. The initialization, control and read-out of this quantum simulator can be accomplished with nitrogen-vacancy centers implanted in diamond. The system can be engineered to simulate a wide variety of interesting strongly-correlated models with long-range dipole-dipole interactions. Due to the superior coherence time of nuclear spins and nitrogen-vacancy centers in diamond, our proposal offers new opportunities towards large-scale quantum simulation at room temperatures

Genetic and polygenic risk score analysis for Alzheimer's disease in the Chinese population

Introduction: Dozens of Alzheimer's disease (AD)-associated loci have been identified in European-descent populations, but their effects have not been thoroughly investigated in the Hong Kong Chinese population. Methods: TaqMan array genotyping was performed for known AD-associated variants in a Hong Kong Chinese cohort. Regression analysis was conducted to study the associations of variants with AD-associated traits and biomarkers. Lasso regression was applied to establish a polygenic risk score (PRS) model for AD risk prediction. Results: SORL1 is associated with AD in the Hong Kong Chinese population. Meta-analysis corroborates the AD-protective effect of the SORL1 rs11218343 C allele. The PRS is developed and associated with AD risk, cognitive status, and AD-related endophenotypes. TREM2 H157Y might influence the amyloid beta 42/40 ratio and levels of immune-associated proteins in plasma. Discussion: SORL1 is associated with AD in the Hong Kong Chinese population. The PRS model can predict AD risk and cognitive status in this population