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

Spread of Mutant Middle East Respiratory Syndrome Coronavirus with Reduced Affinity to Human CD26 during the South Korean Outbreak

The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe respiratory infection with a high mortality rate (similar to 35%). MERS-CoV has been a global threat due to continuous outbreaks in the Arabian peninsula and international spread by infected travelers since 2012. From May to July 2015, a large outbreak initiated by an infected traveler from the Arabian peninsula swept South Korea and resulted in 186 confirmed cases with 38 deaths (case fatality rate, 20.4%). Here, we show the rapid emergence and spread of a mutant MERS-CoV with reduced affinity to the human CD26 receptor during the South Korean outbreak. We isolated 13 new viral genomes from 14 infected patients treated at a hospital and found that 12 of these genomes possess a point mutation in the receptor-binding domain (RBD) of viral spike (S) protein. Specifically, 11 of these genomes have an I529T mutation in RBD, and 1 has a D510G mutation. Strikingly, both mutations result in reduced affinity of RBD to human CD26 compared to wild-type RBD, as measured by surface plasmon resonance analysis and cellular binding assay. Additionally, pseudotyped virus bearing an I529T mutation in S protein showed reduced entry into host cells compared to virus with wild-type S protein. These unexpected findings suggest that MERS-CoV adaptation during human-to-human spread may be driven by host immunological pressure such as neutralizing antibodies, resulting in reduced affinity to host receptor, and thereby impairs viral fitness and virulence, rather than positive selection for a better affinity to CD26. IMPORTANCE Recently, a large outbreak initiated by an MERS-CoV-infected traveler from the Middle East swept South Korea and resulted in 186 confirmed cases with 38 deaths. This is the largest outbreak outside the Middle East, and it raised strong concerns about the possible emergence of MERS-CoV mutations. Here, we isolated 13 new viral genomes and found that 12 of them possess a point mutation in the receptor-binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, CD26, compared to the wild-type virus. These unexpected findings suggest that MERS-CoV adaptation in humans may be driven by host immunological pressure.111819Ysciescopu

Novel Protein Kinase Signaling Systems Regulating Lifespan Identified by Small Molecule Library Screening Using Drosophila

Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity

Hepatitis B vaccinations among Koreans: Results from 2005 Korea National Cancer Screening Survey

<p>Abstract</p> <p>Background</p> <p>Liver cancer is one of most commonly diagnosed cancers among Koreans. Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cancer. HBV infection can be prevented by effective screening and vaccination programs. The purpose of this study is to examine the status of HBV infection and the predictors associated with HBV vaccination.</p> <p>Methods</p> <p>The study population was derived from the 2005 Korea National Cancer Screening Survey (KNCSS). The KNCSS is an annual cross-sectional survey that uses a nationally-representative random sampling to investigate cancer screening rates. A total of 1,786 Koreans over 40 years of age participated in this study.</p> <p>Results</p> <p>Of all the participants, 5.9% reported HBV positive (HBsAg+, HBsAb-), 41.8% were HBV negative but protected (HBsAg-, HBsAb+), and 52.3% were unprotected (HBsAg-, HBsAb-). Among unprotected individuals (n = 934), 23.1% reported to have received the vaccination. About half of those who had vaccinations completed the 3-shot vaccine series. In multiple analyses, education, having private cancer insurance, alcohol use, having regular check-up, and doing regular exercise were associated with completed HBV vaccination.</p> <p>Conclusion</p> <p>This study result suggests that we need a liver cancer education program to increase HBV awareness and to increase the liver cancer prevention message among low educated populations.</p