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  • H together with T also forms


    H109, together with T112, also forms a binding site for H+ (Chen et al., 2004; Harvey et al., 1999). H+-binding at this site also inhibits GlyR-mediated currents. Notably, the vicinity of neurons experiences changes in pH, to a mild extent in physiological conditions such as when neurons fire (Chesler and Kaila, 1992) or to a more severe extent (up to a 1-unit drop in pH) in pathological conditions such as seizure, ischemia and stroke (von Hanwehr et al., 1986). Given its roles in both Zn2+- and H+-binding, the H109 residue affects the function of the GlyR in both physiological and pathological conditions. Previous research shows that the H109A mutation affects GlyR-mediated currents by compromising Zn2+- and H+-binding. However, whether this mutation directly affects the conformation of the remotely-located channel pore is not yet known. In this research, we employ the inhibitory potencies of channel blockers of various molecular volumes as reporters of channel pore conformations. The channel blockers used were niflumic im 12 (NA), picrotoxin (PTX), bilobalide (BB), ginkgolide A (GA), ginkgolide B (GB) and ginkgolide C (GC) (Fig. 2). Previous research from different perspectives demonstrates that each of these compounds inhibits the GlyR current in manners typical of channel blockers. Electrophysiological recordings show that NA's inhibition is voltage-dependent (Maleeva et al., 2017), whereas the inhibition of BB, GA, GB and GC is use-dependent and non-competitive (Hawthorne et al., 2006a; Ivic et al., 2003; Kondratskaya et al., 2002). Further site mutagenesis studies suggest that the inhibition effects of all six compounds are affected by the residues at the lower part of the channel pore, around the M2 2′ and 6′ level (Fig. 1) (Hawthorne et al., 2006b; Shan et al., 2001), implying that these compounds might share the same binding site at this level of the channel pore. In addition, PTX binding to this level in the channel pore has also been verified in a structure study of the GluCl channel (Hibbs and Gouaux, 2011). In this research, we found that their inhibitory potencies were mostly reduced by the H109A mutation and the extents of reduction were positively correlated with the molecular volumes of the channel blockers. In addition, we also found that the H109A mutation slowed the blocking and unblocking rates of the blockers. Taken together, we propose that the H109A mutation might cause some type of conformational change at the blocker binding site, such as a “narrowing” of the channel pore, although other types of conformational change cannot be excluded. This also provides evidence that the H109 residue might allosterically control the channel pore conformation, and Zn2+- or H+-binding at this site might also alter the conformation of the channel pore.
    Material and methods
    Author contributions
    Funding This study was supported by research grants from the National Natural Science Foundation of China (81671361 to Q.S.; 81601135 to L.H.); Department of Education, Guangdong Government under the Top-tier University Development Scheme for Research and Control of Infectious Diseases; Zhejiang Provincial Natural Science Foundation of China (LQ15C090003 to L.H.); Li Ka Shing Foundation.
    Introduction β-cell decline and functional failure play important roles in the development of diabetes mellitus (DM) [1]. β-cells are more sensitive than other types of cells to oxidative stress due to their low levels of antioxidant enzyme expression and activity, and consequently, inner-mitochondrial oxidative stress is the primary pathogenic mechanism of β-cell injury [2]. In DM [3], [4] and in a variety of other diseases [5], activation of NADPH oxidase (also known as Nox) can increase the production of reactive oxygen species (ROS), which induces an oxidative stress state. The NADPH oxidase subtype, Nox2 [6], [7] causes apoptosis of β-cells under high glucose conditions [6], [8], [9], [10]. Thus, inhibiting Nox2 activation and reducing ROS production are viable approaches for protecting islet β-cells and treating diabetes [11].