Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GABAA receptor and nicotinic acetylcholine receptor. Using photo-affinity labeling and coarse-grained molecular dynamics simulations, we identified two fatty acid binding sites in the outer transmembrane domain (TMD) of ELIC. Fatty acid binding to the photolabeled sites is selective for DHA over palmitic acid, and specific for an agonist-bound state. Hexadecyl-methanethiosulfonate modification of one of the two fatty acid binding sites in the outer TMD recapitulates the inhibitory effect of PUFAs in ELIC. The results demonstrate that DHA selectively binds to multiple sites in the outer TMD of ELIC, but that state-dependent binding to a single intrasubunit site mediates DHA inhibition of ELIC.
Figure 4- source data 1 contains the numerical data used to generate Figure 4A and 4B. Figure 4- source data 2 contains the numerical data used to generate Figure 4C and Figure 4- figure supplement 5. Figure 4- source data 3 contains the statistical analysis (linear mixed effects model) for Figure 4- figure supplement 5.
- Wayland WL Cheng
- John T Petroff
- Douglas F Covey
- Douglas F Covey
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Marcel P Goldschen-Ohm, University of Texas at Austin, United States
© 2022, Dietzen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Antioxidant intervention is considered to inhibit reactive oxygen species (ROS) and alleviates hyperglycemia. Paradoxically, moderate exercise can produce ROS to improve diabetes. The exact redox mechanism of these two different approaches remains largely unclear. Here, by comparing exercise and antioxidants intervention on type 2 diabetic rats, we found moderate exercise upregulated compensatory antioxidant capability and reached a higher level of redox balance in the liver. In contrast, antioxidant intervention achieved a low-level redox balance by inhibiting oxidative stress. Both of these two interventions could promote glucose catabolism and inhibit gluconeogenesis through activation of hepatic AMPK signaling, therefore ameliorating diabetes. During exercise, different levels of ROS generated by exercise have differential regulations on the activity and expression of hepatic AMPK. Moderate exercise-derived ROS promoted hepatic AMPK glutathionylation activation. However, excessive exercise increased oxidative damage and inhibited the activity and expression of AMPK. Overall, our results illustrate that both exercise and antioxidant intervention improve blood glucose in diabetes by promoting redox balance, despite different levels of redox balance. These results indicate that the AMPK signaling activation, combined with oxidative damage markers, could act as a sensitive biomarker, reflecting the threshold of redox balance defining effective treatment in diabetes. These findings provide theoretical evidence for the precise treatment of diabetes by antioxidants and exercise.
Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'Site #1', can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'Site #2', that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.