Human ether-à-go-go–related gene (hERG, Kv11.1) potassium channels have unusually slow activation and deactivation kinetics. It has been suggested that, in fast-activating Shaker channels, a highly conserved Phe residue (F290) in the S2 segment forms a putative gating charge transfer center that interacts with S4 gating charges, i.e., R362 (R1) and K374 (K5), and catalyzes their movement across the focused electric field. F290 is conserved in hERG (F463), but the relevant residues in the hERG S4 are reversed, i.e., K525 (K1) and R537 (R5), and there is an extra positive charge adjacent to R537 (i.e., K538). We have examined whether hERG channels possess a transfer center similar to that described in Shaker and if these S4 charge differences contribute to slow gating in hERG channels. Of five hERG F463 hydrophobic substitutions tested, F463W and F463Y shifted the conductance–voltage (G-V) relationship to more depolarized potentials and dramatically slowed channel activation. With the S4 residue reversals (i.e., K525, R537) taken into account, the closed state stabilization by F463W is consistent with a role for F463 that is similar to that described for F290 in Shaker. As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state. However, hERG R537K did not stabilize the open state as predicted. Instead, we found the neighboring K538 residue to be critical for open state stabilization, as K538R dramatically slowed and right-shifted the voltage dependence of activation. Finally, double mutant cycle analysis on the G-V curves of F463W/K525R and F463W/K538R double mutations suggests that F463 forms functional interactions with K525 and K538 in the S4 segment. Collectively, these data suggest a role for F463 in mediating closed–open equilibria, similar to that proposed for F290 in Shaker channels.

A practical and efficient enantioselective synthesis of the calcitonin gene-related peptide receptor antagonist 1 has been developed. The key structural component of the active pharmaceutical ingredient is a syn-1,2-amino-fluoropiperidine 4. Two approaches were developed to synthesize this important pharmacophore. Initially, Ru-catalyzed asymmetric hydrogenation of fluoride-substituted enamide 8 enabled the synthesis of sufficient quantities of compound 1 to support early preclinical studies. Subsequently, a novel, cost-effective route to this intermediate was developed utilizing a dynamic kinetic asymmetric transamination of ketone 9. This synthesis also features a robust Ullmann coupling to install a bis-aryl ether using a soluble Cu(I) catalyst. Finally, an enzymatic desymmetrization of meso-diester 7 was exploited for the construction of the γ-lactam moiety in 1.

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Kv2.1 channels exhibit a U-shaped voltage-dependence of inactivation that is thought to represent preferential inactivation from preopen closed states. However, the molecular mechanisms underlying so-called U-type inactivation are unknown. We have performed a cysteine scan of the S3-S4 and S5-P-loop linkers and found sites that are important for U-type inactivation. In the S5-P-loop linker, U-type inactivation was preserved in all mutant channels except E352C. This mutation, but not E352Q, abolished closed-state inactivation while preserving open-state inactivation, resulting in a loss of the U-shaped voltage profile. The reducing agent DTT, as well as the C232V mutation in S2, restored U-type inactivation to the E352C mutant, which suggests that residues 352C and C232 may interact to prevent U-type inactivation. The R289C mutation, in the S3-S4 linker, also reduced U-type inactivation. In this case, DTT had little effect but application of MTSET restored wild-type-like U-type inactivation behavior, suggestive of the importance of charge at this site. Kinetic modeling suggests that the E352C and R289C inactivation phenotypes largely resulted from reductions in the rate constants for transitions from closed to inactivated states. The data indicate that specific residues within the S3-S4 and S5-P-loop linkers may play important roles in Kv2.1 U-type inactivation.

Lipid metabolic disorders, oxidative stress and inflammation in the liver are key steps in the progression of non-alcoholic fatty liver disease (NAFLD). Ophiopogonin D (OP-D), the main active ingredient of Ophiopogon japonicus , exhibits several pharmacological activities such as antioxidant and anti-inflammatory activities. Therefore, the current study aimed to explore the role of OP-D in NAFLD in a high-fat diet (HFD)-induced obesity mouse model. To investigate the effect of OP-D on NAFLD in vivo , a NAFLD mouse model was established following feeding mice with HFD, then the mice were randomly treated with HFD or HFD + OP-D for 4 weeks. Subsequently, primary mouse hepatocytes were isolated, and enzyme-linked immunosorbent assay, reverse transcription-quantitative PCR western blotting and immunofluorescence analysis were used for assessment to explore the direct effect of OP-D in vitro . The results of the present study indicated that OP-D could ameliorate NAFLD in HFD-induced obese mice by regulating lipid metabolism and antioxidant and anti-inflammatory responses. Additionally, OP-D treatment decreased lipogenesis and inflammation levels in vitro , suggesting that the NF-κB signaling pathway may be involved in the beneficial effects of OP-D on NAFLD.