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

Structural determinants of 5-HT2B receptor activation and biased agonism

Serotonin (5-hydroxytryptamine; 5-HT) receptors modulate a variety of physiological processes ranging from perception, cognition and emotion to vascular and smooth muscle contraction, platelet aggregation, gastrointestinal function and reproduction. Drugs that interact with 5-HT receptors effectively treat diseases as diverse as migraine headaches, depression and obesity. Here we present four structures of a prototypical serotonin receptor—the human 5-HT2B receptor—in complex with chemically and pharmacologically diverse drugs, including methysergide, methylergonovine, lisuride and LY266097. A detailed analysis of these structures complemented by comprehensive interrogation of signaling illuminated key structural determinants essential for activation. Additional structure-guided mutagenesis experiments revealed binding pocket residues that were essential for agonist-mediated biased signaling and β-arrestin2 translocation. Given the importance of 5-HT receptors for a large number of therapeutic indications, insights derived from these studies should accelerate the design of safer and more effective medications. © 2018, The Author(s)

Economic Valuation for Cost–Benefit Analysis of Health Risk Reduction in Drinking Water Systems

Microbial risk mitigation measures in drinking water systems aiming at preventing gastrointestinal disease can provide substantial societal health benefits if implemented properly. However, the procedure of including and monetising the health benefits in cost–benefit analysis (CBA) has been somewhat scattered and inconsistent in the literature, and there is a need for a comparison of available methods. First, through a literature review, we identified the methods to include health benefits in decision support and to monetise these benefits in CBA. Second, we applied the identified health valuation methods in a case study. In the case study, we investigated if changing the health valuation method could change the rank order of the decision alternatives’ net present values. In the case study a risk-based decision model that combined quantitative microbial risk assessment and CBA was used. Seven health valuation methods were identified, each of them including different aspects of health benefits. The results of the case study showed that the choice of the health valuation method can change the rank order of decision alternatives with respect to their net present values. These results highlight the importance of choosing an appropriate health valuation method for the specific application. Although this study focused on the drinking water context, the identified health valuation methods can be applied in any decision support context, provided that input in terms of the health risk reduction is available