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.
100-250 amino acid 'micropeptides' are hidden throughout mammalian genomes, though few functional studies of micropeptides are published. Here, we describe a micropeptide known as the Plasticity Associated Neural Transcript Short (Pants), located in the 22q11.2 region of the human genome, the microdeletion of which conveys a high risk for schizophrenia. Our data show that Pants is upregulated in early adulthood in the mossy fiber circuit of the hippocampus, where it exerts a powerful negative effect on long-term potentiation (LTP). Pants is secreted from neurons, where it associates with synapses but is rapidly degraded with stimulation. Pants dynamically interacts with Rtn4/Nogo, a well-studied regulator of adult plasticity. This study shows that neural micropeptides can act as architectural modules that increase the diversity of the known proteome.
The bestrophin family of calcium-activated chloride channels are integral to eye physiology. Normal function of the human Best1 channel (hBest1) is required for the maintenance of retinal function, and over 250 mutations in the BEST1 gene lead to a collection of related eye diseases broadly termed "bestrophinopathies." The Best2 homolog is expressed in the basolateral membrane of the nonpigmented epithelial cells of the ciliary body, where it has been suggested to play a key role in aqueous humor production through its calcium-dependent activity. Structure-function studies on bestrophins have revealed their overall architecture and established the critical function of two narrow occlusions to the anion conduction pathway, the neck and the aperture. Despite previous studies, questions remain regarding the molecular mechanisms by which mammalian bestrophins maintain their anion selectivity while gating to facilitate ion flow through the channel and across the plasma membrane. This study utilized single-particle cryogenic electron microscopy (CryoEM) to assess conformational dynamics associated with gating in mammalian bestrophin channels. The structures of mammalian bestrophins in various gating states were solved at resolutions ranging from 1.9 to 3.0 Å under different calcium concentrations, providing the molecular basis for proposed gating models. These gating models were then tested by electrophysiological analysis of rationally designed mutants. Our studies provide significant insight into the molecular underpinnings of the biomedically-relevant mammalian bestrophin channels and shed new light into gating dynamics and conserved mechanisms of anion selectivity.
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