Kinetic Model for NS1643 Drug Activation of WT and L529I Variants of Kv11.1 (hERG1) Potassium Channel

Congenital and acquired (drug-induced) forms of the human long-QT syndrome are associated with alterations in Kv11.1 (hERG) channel-controlled repolarizing I_Kr currents of cardiac action potentials. A mandatory drug screen implemented by many countries led to a discovery of a large group of small molecules that can activate hERG currents and thus may act as potent antiarrhythmic agents. Despite significant progress in identification of channel activators, little is known about their mechanism of action. A combination of electrophysiological studies with molecular and kinetic modeling was used to examine the mechanism of a model activator (NS1643) action on the hERG channel and its L529I mutant. The L529I mutant has gating dynamics similar to that of wild-type while its response to application of NS1643 is markedly different. We propose a mechanism compatible with experiments in which the model activator binds to the closed (C3) and open states (O). We suggest that NS1643 is affecting early gating transitions, probably during movements of the voltage sensor that precede the opening of the activation gate.     

Novel roles for hERG K+ channels in cell proliferation and apoptosis

The human ether-a-go-go-related gene potassium channel (hERG, Kv11.1, KCNH2) has an essential role in cardiac action potential repolarization. Electrical dysfunction of the voltage-sensitive ion channel is associated with potentially lethal ventricular arrhythmias in humans. hERG K⁺ channels are also expressed in a variety of cancer cells where they control cell proliferation and apoptosis. In this review, we discuss molecular mechanisms of hERG-associated cell cycle regulation and cell death. In addition, the significance of hERG K⁺ channels as future drug target in anticancer therapy is highlighted.     

hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

Background

The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.

Principal Findings

The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC₅₀ of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca²⁺ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process.

Conclusions/Significance

Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.     

The potassium ion channel opener NS1619 inhibits proliferation and induces apoptosis in A2780 ovarian cancer cells

Diverse types of voltage-gated potassium (K⁺) channels have been shown to be involved in regulation of cell proliferation. The maxi-conductance Ca²⁺-activated K⁺ channels (BK channels) may play an important role in the progression of human cancer. To explore the role of BK channels in regulation of apoptosis in human ovarian cancer cells, the effects of the specific BK channel activator NS1619 on induction of apoptosis in A2780 cells were observed. Following treatment with NS1619, cell proliferation was measured by MTT assay. Apoptosis of A2780 cells pretreated with NS1619 was detected by agarose gel electrophoresis of cellular DNA and flow cytometry. Our data demonstrate that NS1619 inhibits the proliferation of A2780 cells in a dosage and time dependent manner IC₅₀ = 31.1 μM, for 48 h pretreatment and induces apoptosis. Western blot analyses showed that the anti-proliferation effect of NS1619 was associated with increased expression of p53, p21, and Bax. These results indicate that BK channels play an important role in regulating proliferation of human ovarian cancer cells and may induce apoptosis through induction of p21^Cip1 expression in a p53-dependent manner.     

NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V_1/2 of activation of L529I (−34 ± 4 mV) is similar to wild-type (WT) (−37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V_1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (−131 ± 4 mV for K525C and −120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.     

Isoform-Specific Dominant-Negative Effects Associated with hERG1 G628S Mutation in Long QT Syndrome

Background

Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K⁺ channel with properties similar to the rapidly activating delayed rectifying K⁺ current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a_USO isoforms.

Methods

A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation.

Conclusions

The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a_USO isoform. hERG1a_USO-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.     

hERG1 and HIF-2α Behave as Biomarkers of Positive Response to Bevacizumab in Metastatic Colorectal Cancer Patients

Background: In search of novel biomarkers of response to bevacizumab in metastatic colorectal cancer (mCRC), we analyzed the expression and prognostic role of several proteins related to angiogenesis. Methods: A retrospective, multicenter study on 80 surgical samples from mCRC patients treated in first line with bevacizumab plus chemotherapy was accomplished. The following proteins were analyzed by immunohistochemistry: hERG1 potassium channel, β1-integrin, pAKT, NFkB, HIF-1α, HIF-2α, p53, VEGF-A, GLUT-1, and CA-IX. Data were analyzed in conjunction with the clinicopathological characteristics of the patients, KRAS status, response to bevacizumab, and follow-up. Results: (1) All the proteins were expressed in the samples, with statistically significant associations between HIF-1α and gender, HIF-2α and left colon, hERG1 and VEGF-A, β1-integrin and HIF-2α, GLUT-1 and both HIF-1α and HIF-2α, and CA-IX and VEGF-A. (2) At the univariate analysis, positivity for hERG1, VEGF-A, and the active form of HIF-2α (aHIF-2α), and the G3 histological grade showed a positive impact on progression-free survival (PFS). (3) hERG1 and aHIF-2α maintained their positive impact on PFS at the multivariate analysis. (4) hERG1 behaved as a protective factor for PFS independently on KRAS status. Conclusions: hERG1 and aHIF-2α might help to identify patients who would benefit from bevacizumab treatment.     

Up-Regulation of hERG K+ Channels by B-RAF

Human ether-a-go-go related-gene K⁺ channels (hERG) participate in the regulation of tumor cell proliferation and apoptosis. HERG channel activity is up-regulated by growth factors. Kinases sensitive to growth factor signaling include the serine/threonine protein kinase B-RAF. The present study thus explored whether B-RAF influences hERG channel expression and activity. To this end, hERG channels were expressed in Xenopus oocytes with or without wild-type B-RAF, hERG channel activity was determined utilizing dual-electrode voltage clamp and hERG protein abundance in the cell membrane was analyzed utilizing confocal microscopy as well as chemiluminescence. Moreover, in rhabdomyosarcoma RD cells the effect of B-RAF inhibitor PLX-4720 on hERG-mediated current was quantified by whole-cell patch clamp and hERG cell surface protein abundance by utilizing biotinylation of cell surface proteins as well as flow cytometry. As a result, co-expression of wild-type B-RAF in hERG-expressing Xenopus oocytes significantly increased hERG channel activity and hERG channel protein abundance in the cell membrane. Treatment for 24 hours of B-RAF and hERG-expressing Xenopus oocytes with B-RAF inhibitor PLX-4720 (10 µM) significantly decreased hERG-mediated current and hERG cell surface expression. Similarly, in rhabdomyosarcoma RD cells, treatment for 24 hours with B-RAF inhibitor PLX-4720 significantly decreased hERG cell membrane protein abundance and hERG-mediated current. In conclusion, B-RAF is a powerful regulator of hERG channel activity and cell surface hERG protein abundance.     

Rab11-dependent Recycling of the Human Ether-a-go-go-related Gene (hERG) Channel

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (I_Kr). A reduction in the hERG current causes long QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. We reported previously that hERG channels in the plasma membrane undergo vigorous internalization under low K⁺ conditions. In the present study, we addressed whether hERG internalization occurs under normal K⁺ conditions and whether/how internalized channels are recycled back to the plasma membrane. Using patch clamp, Western blot, and confocal imaging analyses, we demonstrated that internalized hERG channels can effectively recycle back to the plasma membrane. Low K⁺-enhanced hERG internalization is accompanied by an increased rate of hERG recovery in the plasma membrane upon reculture following proteinase K-mediated clearance of cell-surface proteins. The increased recovery rate is not due to enhanced protein synthesis, as hERG mRNA expression was not altered by low K⁺ exposure, and the increased recovery was observed in the presence of the protein biosynthesis inhibitor cycloheximide. GTPase Rab11, but not Rab4, is involved in the recycling of hERG channels. Interfering with Rab11 function not only delayed hERG recovery in cells after exposure to low K⁺ medium but also decreased hERG expression and function in cells under normal culture conditions. We concluded that the recycling pathway plays an important role in the homeostasis of plasma membrane-bound hERG channels.     

Transfer of Rolf S3-S4 Linker to hERG Eliminates Activation Gating but Spares Inactivation

Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole.     

Effects of the Small Molecule HERG Activator NS1643 on Kv11.3 Channels

NS1643 is one of the small molecule HERG (Kv11.1) channel activators and has also been found to increase erg2 (Kv11.2) currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3) channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na⁺ or Ca²⁺. At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration.     

Hsp40 Chaperones Promote Degradation of the hERG Potassium Channel

Loss of function mutations in the hERG (human ether-a-go-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.     

hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.     

The voltage-sensing domain of a hERG1 mutant is a cation-selective channel

A cationic leak current known as an “omega current” may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li⁺, K⁺, Cs⁺, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.     

Effects of Tannic Acid,Green Tea and Red Wine on hERG Channels Expressed in HEK293 Cells

Tannic acid presents in varying concentrations in plant foods, and in relatively high concentrations in green teas and red wines. Human ether-à-go-go-related gene (hERG) channels expressed in multiple tissues (e.g. heart, neurons, smooth muscle and cancer cells), and play important roles in modulating cardiac action potential repolarization and tumor cell biology. The present study investigated the effects of tannic acid, green teas and red wines on hERG currents. The effects of tannic acid, teas and red wines on hERG currents stably transfected in HEK293 cells were studied with a perforated patch clamp technique. In this study, we demonstrated that tannic acid inhibited hERG currents with an IC₅₀ of 3.4 μM and ~100% inhibition at higher concentrations, and significantly shifted the voltage dependent activation to more positive potentials (Δ23.2 mV). Remarkably, a 100-fold dilution of multiple types of tea (green tea, oolong tea and black tea) or red wine inhibited hERG currents by ~90%, and significantly shifted the voltage dependent activation to more positive potentials (Δ30.8 mV and Δ26.0 mV, respectively). Green tea Lung Ching and red wine inhibited hERG currents, with IC₅₀ of 0.04% and 0.19%, respectively. The effects of tannic acid, teas and red wine on hERG currents were irreversible. These results suggest tannic acid is a novel hERG channel blocker and consequently provide a new mechanistic evidence for understanding the effects of tannic acid. They also revealed the potential pharmacological basis of tea- and red wine-induced biology activities.     

Interactions of H562 in the S5 Helix with T618 and S621 in the Pore Helix Are Important Determinants of hERG1 Potassium Channel Structure and Function

hERG1 is a member of the cyclic nucleotide binding domain family of K⁺ channels. Alignment of cyclic nucleotide binding domain channels revealed an evolutionary conserved sequence HwX(A/G)C in the S5 domain. We reasoned that histidine 562 in hERG1 could play an important structure-function role. To explore this role, we created in silica models of the hERG1 pore domain based on the KvAP crystal structure with Rosetta-membrane modeling and molecular-dynamics simulations. Simulations indicate that the H562 residue in the S5 helix spans the gap between the S5 helix and the pore helix, stabilizing the pore domain, and that mutation at the H562 residue leads to a disruption of the hydrogen bonding to T618 and S621, resulting in distortion of the selectivity filter. Analysis of the simulated point mutations at positions 562/618/621 showed that the reciprocal double mutations H562W/T618I would partially restore the orientation of the 562 residue. Matching hydrophobic interactions between mutated W562 residue and I618 partially compensate for the disrupted hydrogen bonding. Complementary in vitro electrophysiological studies confirmed the results of the molecular-dynamics simulations on single mutations at positions 562, 618, and 621. Experimentally, mutations of the H562 to tryptophan produced a functional channel, but with slowed deactivation and shifted V_1/2 of activation. Furthermore, the double mutation T618I/H562W rescued the defects seen in activation, deactivation, and potassium selectivity seen with the H562W mutation. In conclusion, interactions between H562 in the S5 helix and amino acids in the pore helix are important determinants of hERG1 potassium channel function, as confirmed by theory and experiment.     

The F-box protein SKP2 mediates androgen control of p27 stability in LNCaP human prostate cancer cells

Background

The cyclin-dependent kinase inhibitor p27 is a putative tumor suppressor that is downregulated in the majority of human prostate cancers. The mechanism of p27 down-regulation in prostate cancers in unknown, but presumably involves increased proteolysis mediated by the SCF^SKP2 ubiquitin ligase complex. Here we used the human prostate cancer cell line LNCaP, which undergoes G1 cell cycle arrest in response to androgen, to examine the role of the SKP2 F-box protein in p27 regulation in prostate cancer.

Results

We show that androgen-induced G1 cell cycle arrest of LNCaP cells coincides with inhibition of cyclin-dependent kinase 2 activity and p27 accumulation caused by reduced p27 ubiquitylation activity. At the same time, androgen decreased expression of SKP2, but did not affect other components of SCF^SKP2. Adenovirus-mediated overexpression of SKP2 led to ectopic down-regulation of p27 in asynchronous cells. Furthermore, SKP2 overexpression was sufficient to overcome p27 accumulation in androgen arrested cells by stimulating cellular p27 ubiquitylation activity. This resulted in transient activation of CDK2 activity, but was insufficient to override the androgen-induced G1 block.

Conclusions

Our studies suggest that SKP2 is a major determinant of p27 levels in human prostate cancer cells. Based on our in vitro studies, we suggest that overexpression of SKP2 may be one of the mechanisms that allow prostate cancer cells to escape growth control mediated by p27. Consequently, the SKP2 pathway may be a suitable target for novel prostate cancer therapies.     

Vacuolar ATPase driven potassium transport in highly metastatic breast cancer cells

Breast cancer is the second leading cause of death in women and thus has received a great deal of attention by researchers. Recent studies suggested decreased occurrence of cancer in patients treated with cardiac glycosides (CGs) for heart conditions. Because CGs induce their cellular effects via the Na⁺, K⁺ ATPase (Na–K), we treated four breast cancer cell lines (MCF-7, T47D, MDA-MB453, and MDA-MB231) and a non-cancerous breast ductal epithelial cell line (MCF-10A) with ouabain, a well-characterized CG, and measured cell proliferation by measuring bromodeoxyuridine incorporation. Ouabain (1 μM) decreased cell proliferation in all cell lines studied except MDA-MB453 cells. Western blot of Na–K α and β subunits showed α1, α3, and β1 expression in all cell lines except MDA-MB453 cells where Na–K protein and mRNA were absent. Potassium uptake, measured as rubidium (⁸⁶Rb) flux, and intracellular potassium were both significantly higher in MDA-MB453 cells compared to MCF-10A cells. RT-qPCR suggested a 7 fold increase in voltage-gated potassium channel (KCNQ2) expression in MDA-MB453 cells compared to MCF-10A cells. Inhibition of KCNQ2 prevented cell growth and ⁸⁶Rb uptake in MDA-MB453 cells but not in MCF-10A cells. All cancer cells had significantly higher vacuolar H-ATPase (V-ATPase) activity than MCF-10A cells. Inhibition of V-ATPase decreased ⁸⁶Rb uptake and intracellular potassium in MDA-MB453 cells but not in MCF-10A cells. The findings point to the absence of Na–K, high hERG and KCNQ2 expression, elevated V-ATPase activity and sensitivity to V-ATPase inhibitors in MDA-MB453. We conclude that cancer cells exhibit fundamentally different metabolic pathways for maintenance of intracellular ion homeostasis.     

The solution structure of the S4–S5 linker of the hERG potassium channel

The human ether‐à‐go‐go related gene (hERG) encodes a protein that forms a voltage‐gated potassium channel and plays an important role in the heart by controlling the rapid delayed rectifier K⁺ current (I_Kr). The S4–S5 linker was shown to be important for the gating of the hERG channel. Nuclear magnetic resonance study showed that a peptide derived from the S4–S5 linker had no well‐ordered structure in aqueous solution and adopted a 3₁₀‐helix (E544‐Y545‐G546) structure in detergent micelles. The existence of an amphipathic helix was confirmed, which may be important for interaction with cell membrane. Close contact between side chains of residues R541 and E544 was observed, which may be important for its regulation of channel gating. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Overexpression of p75NTR increases survival of breast cancer cells through p21waf1

The p75 neurotrophin receptor (p75^NTR) plays a critical role in various neuronal and non-neuronal cell types by regulating cell survival, differentiation and proliferation. To evaluate the influence of p75^NTR in breast cancer development, we have established and characterized breast cancer cells which stably overexpress p75^NTR. We showed that p75^NTR overexpression per se promoted cell survival to apoptogens with a concomitant slowdown of cell growth. The pro-survival effect is associated with an increased expression of the inhibitor of apoptosis protein-1 (c-IAP1), a decrease of TRAIL-induced cleavage of PARP, procaspase 9 and procaspase 3, and a decrease of cytochrome C release from the mitochondria. The anti-proliferative effect is due to a cell accumulation in G0/G1, associated with a decrease of Rb phosphorylation and an increase of p21^waf1. Interestingly, inhibition of p21^waf1 with siRNA not only restores proliferation but also abolishes the pro-survival effect of p75^NTR, indicating the key role of p21^waf1 in the biological functions of p75^NTR. Finally, using a SCID mice xenograft model, we showed that p75^NTR overexpression favors tumor growth and strongly increases tumor resistance to anti-tumoral treatment.Together, our findings suggest that p75^NTR overexpression in breast tumor cells could favor tumor survival and contribute to tumor resistance to drugs. This provides a rationale to consider p75^NTR as a potential target for the future design of innovative therapeutic strategies.