Bestrophin-2 and glutamine synthetase form a complex for glutamate release

Bestrophin-2 (Best2), a member of the bestrophin family of calcium-activated anion channels, is expressed in nonpigmented epithelium (NPE) of the ciliary body with critical involvement in aqueous humor homeostasis. Best2 knockout mice exhibit a reduced intraocular pressure, suggesting the pharmaceutical potential of Best2 as a target for lowering ocular hypertension. However, little is known about the physiological binding partners and/or regulators of Best2. In this study, we performed a proteomic pulldown of bovine ciliary body lysate using purified Best2 proteins as the bait, and identified glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia into glutamine, as a Best2 binding partner. We assessed the functional consequences of the interaction and discovered a mutually inhibitory effect: GS decreases Best2-mediated chloride conduction, while Best2 reduces cytosolic GS activity by tethering GS to the cell membrane. Since glutamate is the substrate of GS and an anion, we investigated the ability of Best2 to conduct glutamate. Remarkably, Best2 exhibits a directional permeability to glutamate heavily favoring glutamate exit from the cell, and the binding of GS to Best2 sensitized Best2 to intracellular glutamate, which promotes opening of Best2 and thus relieves the inhibitory effect of GS. We further demonstrated the physiological role of Best2 in conducting chloride and glutamate and the influence of GS in NPE cells. Moreover, by single-particle cryo-EM, we solved the structure of the Best2-GS complex, illustrating the interacting residues and extension of the Best2 ion conducting pathway through the GS central cavity. Together, our results reveal a novel mechanism of glutamate release through Best2-GS.