Bestrophin-1 (Best1) and bestrophin-2 (Best2) are two members of the bestrophin family of calcium (Ca²⁺)-activated chloride (Cl⁻) channels with critical involvement in ocular physiology and direct pathological relevance. Here, we report cryo-EM structures of wild-type human Best1 and Best2 in various states at up to 1.8 Å resolution. Ca²⁺-bound Best1 structures illustrate partially open conformations at the two Ca²⁺-dependent gates of the channels, in contrast to the fully open conformations observed in Ca²⁺-bound Best2, which is in accord with the significantly smaller currents conducted by Best1 in electrophysiological recordings. Comparison of the closed and open states reveals a C-terminal auto-inhibitory segment (AS), which constricts the channel concentrically by wrapping around the channel periphery in an inter-protomer manner and must be released to allow channel opening. Our results demonstrate that removing the AS from Best1 and Best2 results in truncation mutants with similar activities, while swapping the AS between Best1 and Best2 results in chimeric mutants with swapped activities, underlying a key role of the AS in determining paralog specificity among bestrophins.

In the mammalian glutamate transporters, countertransported intracellular K⁺ is essential for relocating the glutamate binding site to the extracellular side of the membrane. This K⁺-dependent process is believed to be rate limiting for the transport cycle. In contrast, extracellular K⁺ induces glutamate release upon transporter reversal. Here, we analyzed potential K⁺ binding sites using molecular dynamics (MD) simulations and site-directed mutagenesis. Two candidate sites were identified by spontaneous K⁺ binding in MD simulations, one site (K1 site) overlapping with the Na1 Na⁺ binding site and the K2 site being localized under hairpin loop 2 (HP2). Mutations to conserved amino acid residues in these sites resulted in several transporters that were defective in K⁺-induced reverse transport and which bound K⁺ with reduced apparent affinity compared with the wild-type transporter. However, external K⁺ interaction was abolished in only one mutant transporter EAAC1_D454A in the K1 site. Our results, for the first time, directly demonstrate effects of K1-site mutations on K⁺ binding, in contrast to previous reports on K⁺ binding sites based on indirect evidence. We propose that K⁺ binding to the K1 site is responsible for catalyzing the relocation step, whereas binding to the K2 site may have an as-of-yet unidentified regulatory function.

Plasma membrane–associated glutamate transporters play a key role in signaling by the major excitatory neurotransmitter glutamate. Uphill glutamate uptake into cells is energetically driven by coupling to co-transport of three Na⁺ ions. In exchange, one K⁺ ion is counter-transported. Currently accepted transport mechanisms assume that Na⁺ and K⁺ effects are exclusive, resulting from competition of these cations at the binding level. Here, we used electrophysiological analysis to test the effects of K⁺ and Na⁺ on neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1; the rat homologue of human excitatory amino acid transporter 3 (EAAT3)). Unexpectedly, extracellular K⁺ application to EAAC1 induced anion current, but only in the presence of Na⁺. This result could be explained with a K⁺/Na⁺ co-binding state in which the two cations simultaneously bind to the transporter. We obtained further evidence for this co-binding state, and its anion conductance, by analyzing transient currents when Na⁺ was exchanged for K⁺ and effects of the [K⁺]/[Na⁺] ratio on glutamate affinity. Interestingly, we observed the K⁺/Na⁺ co-binding state not only in EAAC1 but also in the subtypes EAAT1 and -2, which, unlike EAAC1, conducted anions in response to K⁺ only. We incorporated these experimental findings in a revised transport mechanism, including the K⁺/Na⁺ co-binding state and the ability of K⁺ to activate anion current. Overall, these results suggest that differentiation between Na⁺ and K⁺ does not occur at the binding level but is conferred by coupling of cation binding to conformational changes. These findings have implications also for other exchangers.

Bestrophin-1 (Best1) is an anion channel genetically linked to vision-threatening retinal degenerative channelopathies. Here, we identify interactions between Best1 and both isoforms of glutamic acid decarboxylases (GAD65 and GAD67), elucidate the distinctive influences of GAD65 and GAD67 on Best1’s permeability to various anions/neurotransmitters, discover the functionality of Best1 as a γ-Aminobutyric acid (GABA) type A receptor, and solve the structure of GABA-bound Best1. GAD65 and GAD67 both promote Best1-mediated Cl⁻ currents, but only GAD65 drastically enhances the permeability of Best1 to glutamate and GABA, for which GAD67 has no effect. GABA binds to Best1 on an extracellular site and stimulates Best1-mediated Cl⁻ currents at the nano-molar concentration level. The physiological role of GAD65 as a cell type-specific binding partner and facilitator of Best1 is demonstrated in retinal pigment epithelial cells. Together, our results reveal critical regulators of Best1 and inform a network of membrane transport metabolons formed between bestrophin channels and glutamate metabolic enzymes.

Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, apotent, non-competitive inhibitor of EAAT1. Alanine serine cysteine transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine between intracellular and extracellular compartments . ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here, we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na⁺ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2. Guided by computational modeling using ligand docking and molecular dynamics simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double-mutant transporter, 100% of the inhibitory effect of UCPH-101 could be restored, and the apparent affinity was increased (K_i = 4.3 μM), much closer to the EAAT1 value of 0.6 μM. Finally, we identify a novel non-competitive ASCT2 inhibitor, through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective ASCT2 allosteric inhibitors can be designed .