Selectivity filter instability dominates the low intrinsic activity of the TWIK-1 K2P K+ channel

Two-pore domain K+ (K2P) channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by posttranslational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating this further, we observed that the low activity of the SF gate appears to arise from the inefficiency of K+ in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb+, NH4+, and Cs+, strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via an SF-mediated gating mechanism, but we found here that only very strong nonphysiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb+ currents are potently inhibited by intracellular K+ (IC50 = 2.8 mM). We conclude that TWIK-1 displays unique SF gating properties among the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K+ appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface

Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions

Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

BACKGROUND:Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs. METHODOLOGY/PRINCIPAL FINDINGS:We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T-->S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding. CONCLUSIONS/SIGNIFICANCE:The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features

Need for a Standardized Translational Drug Development Platform: Lessons Learned from the Repurposing of Drugs for COVID-19

In the absence of drugs to treat or prevent COVID-19, drug repurposing can be a valuable strategy. Despite a substantial number of clinical trials, drug repurposing did not deliver on its promise. While success was observed with some repurposed drugs (e.g., remdesivir, dexamethasone, tocilizumab, baricitinib), others failed to show clinical efficacy. One reason is the lack of clear translational processes based on adequate preclinical profiling before clinical evaluation. Combined with limitations of existing in vitro and in vivo models, there is a need for a systematic approach to urgent antiviral drug development in the context of a global pandemic. We implemented a methodology to test repurposed and experimental drugs to generate robust preclinical evidence for further clinical development. This translational drug development platform comprises in vitro, ex vivo, and in vivo models of SARS-CoV-2, along with pharmacokinetic modeling and simulation approaches to evaluate exposure levels in plasma and target organs. Here, we provide examples of identified repurposed antiviral drugs tested within our multidisciplinary collaboration to highlight lessons learned in urgent antiviral drug development during the COVID-19 pandemic. Our data confirm the importance of assessing in vitro and in vivo potency in multiple assays to boost the translatability of pre-clinical data. The value of pharmacokinetic modeling and simulations for compound prioritization is also discussed. We advocate the need for a standardized translational drug development platform for mild-to-moderate COVID-19 to generate preclinical evidence in support of clinical trials. We propose clear prerequisites for progression of drug candidates for repurposing into clinical trials. Further research is needed to gain a deeper understanding of the scope and limitations of the presented translational drug development platform

Altered and dynamic ion selectivity of K+ channels in cell development and excitability

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The family of K 2P channels: salient structural and functional properties

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Use of mathematical laws for optimizing the dose of swollen and dry bentonite during the fining of white wines. Part II : influence on the volume of lies and economics

A Sauvignon white wine was fined with a dry bentonite (BS), e.g directly incorporated in the wine without swelling treatment, or with the same bentonite swelled in water 24 hours before utilization (BG). The volumes of lees generated by the fining were situated between 0,27 and 1,51 % (v/v) for BS, and between 1,07 and 4,59 % (v/v) for BG. The relation between the quantity of bentonite introduced in the wine (g/hl) and the volume of lees (v/v) follows a power law. The volume of lees only increased by 70 % when the dose of swelled bentonite was doubled; for higher doses of bentonite (rarely used), one can observe a packing of the lees. For BS, lees were approximately twice more volumetric when the dose was doubled. We also observe very good relationship between the clarifying efficiency and the volume of lees. When the clarifying efficiency of BG increased by 10 % the volume of lees increased by 74 %. For BS, when the clarifying efficiency increased by 10 % the volume of lees increased by 86 % because of its low efficiency during clarification. The decrease of the natural proteic haze risk and the volume of lees generated by bentonite fining were also closely correlated by an exponential law. For BG, the mathematical law showed that when the volume of lees increased by 0,1 % (v/v), colloidal haze risk decreased only by 22 %. On the contrary, for BS, when the volume of lees increased by 0,1 %, proteic haze risk decreased by 44 %. Finally, the estimation of the cost of fining showed that the use of a non swelled bentonite was economically more interesting than the utilization of a swelled bentonite. This was true when this Sauvignon wine is sold both in bottle or in bulk. For the studied wine, the estimated winning was 115 euros for 10 hl sold in bulk

Performance of epistasis detection methods in semi-simulated GWAS

International audienceBackground: Part of the missing heritability in Genome Wide Association Studies (GWAS) is expected to be explained by interactions between genetic variants, also called epistasis. Various statistical methods have been developed to detect epistasis in case-control GWAS. These methods face major statistical challenges due to the number of tests required, the complexity of the Linkage Disequilibrium (LD) structure, and the lack of consensus regarding the definition of epistasis. Their limited impact in terms of uncovering new biological knowledge might be explained in part by the limited amount of experimental data available to validate their statistical performances in a realistic GWAS context. In this paper, we introduce a simulation pipeline for generating real scale GWAS data, including epistasis and realistic LD structure. We evaluate five exhaustive bivariate interaction methods, fastepi, GBOOST, SHEsisEpi, DSS, and IndOR. Two hundred thirty four different disease scenarios are considered in extensive simulations. We report the performances of each method in terms of false positive rate control, power, area under the ROC curve (AUC), and computation time using a GPU. Finally we compare the result of each methods on a real GWAS of type 2 diabetes from the Welcome Trust Case Control Consortium. Results: GBOOST, SHEsisEpi and DSS allow a satisfactory control of the false positive rate. fastepi and IndOR present an increase in false positive rate in presence of LD between causal SNPs, with our definition of epistasis. DSS performs best in terms of power and AUC in most scenarios with no or weak LD between causal SNPs. All methods can exhaustively analyze a GWAS with 6.10 5 SNPs and 15,000 samples in a couple of hours using a GPU. Conclusion: This study confirms that computation time is no longer a limiting factor for performing an exhaustive search of epistasis in large GWAS. For this task, using DSS on SNP pairs with limited LD seems to be a good strategy to achieve the best statistical performance. A combination approach using both DSS and GBOOST is supported by the simulation results and the analysis of the WTCCC dataset demonstrated that this approach can detect distinct genes in epistasis. Finally, weak epistasis between common variants will be detectable with existing methods when GWAS of a few tens of thousands cases and controls are available

Use of mathematical laws for optimizing the doses of swelled and dry bentonite during the fining of white wines. Part I: clarification and colloidal stability

White wine clarity is of prime importance for the winemaker as a bottle showing haziness is likely to be rejected by the consumer. It is then important to ensure a perfect colloidal stability to the wine. This study concerns a Sauvignon white wine from the Touraine area (vintage 2000). We have determined the relationships existing between the dose of bentonite used, the manner for preparing the bentonite (the dry cristalites can be directly introduced in the wine; the bentonite can also be used after swelling in water), the decrease of wine protein haze and clarifying efficiency. Clarifying kinetics are identical for 10 g/hl swelled bentonite (SB) and 100 g/hl dry bentonite (DB). The difference of efficiency between SB and DB is all the more marked than the dose used increases. At the end of the kinetics, for identical treatments (the doses are between 10 and 100 g/hl), wines fined with DB have turbidity 2-3 times higher than the same wines fined with the same doses of SB. The mathematical law shows that the turbidity decreases by 17 % when the dose of dry bentonite is doubled. For this example, the relationship between these two parameters follows a power law. The decrease reaches 27 % when the wine is fined with SB. According to heat treatment, the wine must be treated with 30 g/hl SB and 60 g/hl DB to present a good colloidal stability. For this Sauvignon wine, fined with SB, haze risk decreases by 82 % each time the dose of bentonite increases by 10 g/hl. In this case, the relationship between these two parameters follows an exponential law. If an addition of oak tannins is made in the wine fined with 30 g/hl SB or 60 g/hl DB, any trouble appears. For heat treatment test and tannin addition test, the ratio is each time 2 DB for 1 SB to have a correctly fined wine. On the basis of these results, the use of dry bentonite seems to be less interesting than the use of swelled bentonite

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors

International audienceLigand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering