Dissecting the structure and function of bestrophin channels

Abstract

Purpose : Over 200 distinct mutations in the human BEST1 gene have been documented to cause a spectrum of retinal degenerative diseases called bestrophinopathies. BEST1 encodes an ion channel, bestrophin1 (BEST1), which is indispensable for the vision-related Ca²⁺-dependent Cl^- current in retinal pigment epithelium (RPE). Therefore, dissecting the structure and function of the BEST1 channel by mutagenesis is of great value to both basic and clinical studies. However, residue substitution is often intolerable on critical sites, resulting in a complete or severe loss of the channel structure/function and thus yielding little mechanistic information. We aim to rationally predict critical and structurally flexible residues on BEST1, which will allow non-disruptive mutagenesis on the channel for functional and structural analyses.

Methods : Our recently reported bacterial Klebsiella pneumoniae bestrophin (KpBest) structure provides a unique opportunity to address this task, as bestrophin homologs have conserved structures but KpBest and BEST1 share only 14% sequence identity. Hence, the most critical and structurally flexible residues on BEST1 are naturally highlighted in the alignment with KpBest. We studied bestrophins with multidisciplinary approaches including lipid bilayer, patch clamp, microscale thermophoresis and X-ray crystallography.

Results : We identified multiple non-disruptive bestrophin mutants, and discovered that a conserved region in bestrophins is critical for interaction of the channel with a novel activator. Importantly, a patient-derived BEST1 mutation located within the binding motif caused a significant deficiency in channel activation (Fig. A, *p < 0.05 compared to WT in the presence of the activator), a diminished binding affinity to the activator (Fig. B, K_d 147 mM vs.16 mM), and an apparent conformational change in the channel ion conducting pathway.

Conclusions : Our results identified a new and critical activator of bestrophins, and revealed a disease-causing mechanism of BEST1 mutations.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Patient-specific mutations impair BEST1’s essential role in mediating CaCC currents in human RPE. (A) Bar chart showing the endogenous Ca²⁺-dependent Cl^- current amplitudes of WT and mutant RPEs in the absence and presence of 2 mM activator. (B) Microscale thermophoresis binding curves of WT and mutant proteins to the activator.