Polyunsaturated fatty acids inhibit a pentameric ligand-gated ion channel through one of two binding sites.

Mark J Arcario,Noah M Dietzen,Douglas F Covey,K Trent Moreland,Grace Brannigan,Kathiresan Krishnan,John T Petroff,Lawrence J Chen,Wayland WL Cheng

doi:10.7554/elife.74306

Mark J Arcario, Noah M Dietzen + Show 7 more

Open Access

https://doi.org/10.7554/elife.74306

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Abstract

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.

Highlights

Fatty acids are major components of the cell membrane, and modulators of many ion channels including pentameric ligand-gated ion channels [1,2,3,4,5,6]
The effect of fatty acids such as docosahexaenoic acid (DHA) (22:6) was determined by pre-applying fatty acid to the patch for 3 min followed by rapid application of cysteamine with the same concentration of fatty acid used in the pre-application (Figure 1A)
The results suggest that the mechanism of fatty acid inhibition of the GABAA receptor (GABAAR) and nicotinic acetylcholine receptor (nAchR) is present in Erwinia ligand-gated ion channel (ELIC)

Summary

INTRODUCTION

Fatty acids are major components of the cell membrane, and modulators of many ion channels including pentameric ligand-gated ion channels (pLGICs) [1,2,3,4,5,6]. Fatty acids modulate ion channel function allosterically by one of two general mechanisms: by direct binding to specific sites in the protein, or by altering the physical properties of the lipid bilayer and thereby indirectly impacting protein structure. A direct mechanism is thought to be important in pLGICs based on a crystal structure of Gloeobacter ligand-gated ion channel (GLIC) in complex with docosahexaenoic acid (DHA), which showed a single binding site for DHA in the outer portion of the transmembrane domain (TMD) of this channel [3]. Mutagenesis studies and molecular dynamics simulations support a direct mechanism of fatty acid modulation of large conductance calcium-activated potassium channels (BK) [14] and voltagegated potassium channels (Kv) [15,16], but biochemical identification of fatty acid binding sites in these and other ion channels remains a challenge The results argue for a direct mechanism of DHA inhibition of ELIC through state-dependent binding to a single site

RESULTS

DISCUSSION

MATERIALS AND METHODS

723 ACKNOWLEDGEMENTS

Methods