Effect of bestrophin-1 on L-type Ca2+ channel activity depends on the Ca2+ channel beta-subunit.
Best's vitelliforme macular degeneration is an inherited retinal degeneration associated with a reduction of the light-peak in the patient's electro-oculogram. Bestrophin-1, the product of the disease-promoting/forming gene can function as regulator of voltage-dependent L-type Ca(2+) channels in the retinal pigment epithelium (RPE). Since mice deficient for either β4-subunits or Ca(V)1.3 subunits show reduced light-peaks, the regulatory function of bestrophin-1 on heterologously expressed Ca(2+) channels composed of the pore-forming Ca(V)1.3 and the auxiliary β4-subunit was analyzed. Precipitation of β4-subunits led to co-precipitation with bestrophin-1 and subsequent analysis of subcellular localization showed co-localization of bestrophin-1, Ca(V)1.3 and β4-subunit in the cell membrane. Ca(V)1.3 currents in the presence of β4-subunits and bestrophin-1 showed accelerated time-dependent activation and decreased current density compared to currents measured in the absence of bestrophin-1. In the presence of the β3-subunit, which is not expressed in the RPE bestrophin-1 did not modulate Ca(V)1.3 activity. Deletion of a cluster of proline-rich motifs in the C-terminus of bestrophin-1 reduced its co-immuno precipitation with the β4-subunit and strongly reduced the Ca(V)1.3 activity. Cells co-expressing bestrophin-1 lacking the proline-rich motifs and Ca(V)1.3 subunits showed less efficient trafficking of bestrophin-1 into the cell membrane. In summary, we conclude that bestrophin-1 modulates L-type channels of the RPE via proline-rich motif-dependent interaction with β4-subunits. A disturbed interaction reduces the currents of the Ca(V)1.3 subunits. This mechanism could open new ways to understand changes in the patient's electro-oculogram and functional alterations of the RPE leading to retinal degeneration.
Reichhart
Nadine
N
Experimental Ophthalmology, Eye Hospital, University Medical Center Regensburg, Franz-Josef-Strauß Allee 11, 93053 Regensburg, Germany.
Milenkovic
Vladimir M
VM
Halsband
Claire-Amelie
CA
Cordeiro
Sönke
S
Strauss
Olaf
O
D000595:Amino Acid Sequence; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D000075002:Bestrophins; D015153:Blotting, Western; D016466:CHO Cells; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D018118:Chloride Channels / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000502:physiology*; D006801:Humans; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000196:embryology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D055232:Proline-Rich Protein Domains; D011485:Protein Binding; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D014161:Transduction, Genetic
C113133:BEST1 protein, human; D000075002:Bestrophins; C472073:CACNA1D protein, human; C107598:CACNB4 protein, human; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D018118:Chloride Channels; D005136:Eye Proteins