Direct Interaction of Bestrophin-1 and Beta-subunits of Voltage-Dependent Calcium Channels

Abstract

Purpose: : Best’s disease is caused by mutations in the VMD2 gene. Recent publications implied that the VMD2 gene product, bestrophin-1, can combine the function as Cl channel and as modulator of voltage-dependent Ca²⁺ channels. The latter function could explain the changes in the electroretinogram (EOG) of Best patients because the light-peak of the EOG is dependent on the activity of voltage-dependent Ca²⁺ channels. Purpose of the study is to analyze the underlying mechanism how bestrophin-1 modulates Ca²⁺ channels.

Methods: : Heterologeous expression of bestrophin-1 and subunits of voltage-dependent Ca²⁺ channels in CHO or COS-7 cells. Isolation of membrane proteins from fresh human RPE cells. Immunoprecipitation and western-blot analysis of the precipitated proteins.

Results: : CHO cells were used to express bestrophin-1 and Ca²⁺ channel subunits. Western-blot analysis of proteins obtained by precipitation of the pore-forming CaV1.3 subunit revealed the co-immunoprecipitation with the auxiliary beta3-subunit of voltage-dependent Ca²⁺ channels. Preipitation of CaV1.3 subunits did not co-precipitate these subunits with bestrophin-1. However, precipitation of the auxiliary beta3-subunit of voltage-dependent Ca²⁺ channels did result in co-precipitation with bestrophin-1. This was confirmed by precipitation of bestrophin-1. In these precipitates, the beta3-subunit of voltage-dependent Ca²⁺ channels was detected. The same was observed when using COS-7 cells as expression system. Here, also the beta3-subunits could be co-precipitated with bestrophin-1. Furthermore, bestrophin-1 and beta3-subunits could be co-immunoprecipitated from membrane proteins from freshly isolated human RPE cells.

Conclusions: : Using co-immunoprecipitation experiments, the interaction between bestrophin-1 and Ca²⁺ channel subunits was analyzed. The in these experiments we could confirm the known physical interaction between the pore-forming CaV1.3 subunits and the auxiliary beta3-subunits. Independent from the expression system and also in freshly isolated RPE cells we could detect physical interaction between the beta3-subunits of Ca channels and bestrophin-1. This physical interaction corresponds nicely with the influence of bestrophin-1 on voltage-dependent Ca²⁺ channel activity which are characteristic for beta-subunits.

Keywords: ion channels • retinal pigment epithelium • calcium