Interaction of Bestrophin-1 and Ca2+ Channel β-Subunits: Identification of New Binding Domains on the Bestrophin-1 C-Terminus

Abstract

Bestrophin-1 modulates currents through voltage-dependent L-type Ca2+ channels by physically interacting with the β-subunits of Ca2+ channels. The main function of β-subunits is to regulate the number of pore-forming CaV-subunits in the cell membrane and modulate Ca2+ channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca2+ channel subunits, together with modulation of CaV1.3 Ca2+ channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and CaV1.3 subunits partly lost membrane localization. Currents from CaV1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca2+ channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming CaV Ca2+-channel subunits.