Altered L-type Ca²⁺ channel activity by mutant bestrophin-1 in RPE cells

Abstract

Purpose : Mutations in BEST1 cause Best’s vitelliforme macular dystrophy. The BEST1 gene poduct bestrophin-1 regulates the activity of L-type Ca²⁺ channels by direct interaction with their β-subunits. Inhibition or knockout of L-type channels in retinal pigment epithelium (RPE) reduce the electro-oculogram light-peak, hallmark for diagnosis of Best’s disease . Thus, the bestrophin-1/Ca²⁺ channel interaction is of functional relevance for the RPE and BEST1 mutations develop major patho-mechanisms by disturbed interaction.

Methods : CHO cells transfected with Cav1.3 L-type channels and mutant bestrophin-1 (T6P, F80L, R218C, F305S) were used to analyze Ca²⁺ channels by patch-clamp and study protein interaction by immunoprecipitation. Plasma membrane localization was quantified in primary porcine RPE cells and iPS-derived human RPE cells expressing endogenously wildtype bestrophin-1 and heterologeously mutant bestrophin-1.

Results : All four mutant bestrophin-1 directly interacted with L-type channels composed of Cav1.3, β4 subunits at lower immunopcrecipitation efficiency compared to that of wildtype bestrophin-1. Mutant F80L and F305S bestrophin-1 reduced the L-type channel current density and changed voltage-dependence. Reduced current densities correlated with reduced plasma membrane localization in CHO cells of mutant bestrophin-1 and pore-forming subunit Cav1.3. In confluent porcine RPE cells, mutant bestrophin-1 reduced the plasma membrane localization of Cav1.3 channels. In porcine RPE cells, the mutant bestrophin-1 show misplacement in the cell: R218C was less present at the basolateral side; T6P, F80L and F305S were localized in the cytosol; T6P appeared at the apical side. Using GFP-tagged mutant bestrophin-1, we showed that mutant bestrophin-1 inhibits trafficking of wild-type bestrophin-1 as well. The Cav1.3 subunits co-localized with bestrophin-1 but showed reduced plasma membrane expression in the presence of T6P, F80L and F305S. R218C itself shows weakly reduced membrane localization and Cav1.3 mislocalization. Human iPS-derived RPE cells confirmed the data for T6P and R218C.

Conclusions : The trafficking defect of mutant bestrophin-1 reduced the plasma membrane localization of L-type Ca²⁺ channels in the RPE by physical interaction of bestrophin-1 and the Ca²⁺ channels β-subunit. Decreased L-type channel activity would change RPE function and explain reduced light-peak in Best patient’s electro-oculogram.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.