title: 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 Ca(2+) channels by physically interacting with the β-subunits of Ca(2+) channels. The main function of β-subunits is to regulate the number of pore-forming Ca(V)-subunits in the cell membrane and modulate Ca(2+) channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca(2+) channel subunits, together with modulation of Ca(V)1.3 Ca(2+) 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 Ca(V)1.3 subunits partly lost membrane localization. Currents from Ca(V)1.3 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 Ca(2+) 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 Ca(V) Ca(2+)-channel subunits.
authors.affiliation: Experimental Ophthalmology, Eye Hospital, University Medical Center Regensburg, Regensburg, Germany.
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Milenkovic
authors.forename: Sarka
authors.initials: S
authors.lastname: Krejcova
authors.forename: Nadine
authors.initials: N
authors.lastname: Reichhart
authors.forename: Andrea
authors.initials: A
authors.lastname: Wagner
authors.forename: Olaf
authors.initials: O
authors.lastname: Strauss
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000075002:Bestrophins; D016466:CHO Cells; D015220:Calcium Channels / Q000737:chemistry*; D018118:Chloride Channels / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D005136:Eye Proteins / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: C113133:BEST1 protein, human; D000075002:Bestrophins; D015220:Calcium Channels; D018118:Chloride Channels; D005136:Eye Proteins