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Expression of bestrophin-1, the product of the VMD2 gene, modulates voltage-dependent Ca²⁺ channels in retinal pigment epithelial cells

Rita Rosenthal^*,, Benjamin Bakall^,, Tyson Kinnick, Neal Peachey^||,¶, Sönke Wimmers^#, Claes Wadelius, Alan Marmorstein and Olaf Strauss^#,1

^* Augenklinik, Charité-Universitaetsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany;
Institut fuer Klinische Physiologie, Charité-Universitaetsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany;
Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden;
Department of Ophthalmology, University of Arizona College of Medicine, Tucson, Arizona, USA;
^|| Cole Eye Institute, Cleveland Clinic Foundation, Cleveland Ohio, USA;
^¶ Research Service, Cleveland VA Medical Center, Cleveland Ohio, USA; and
^# Experimentelle Ophthalmologie, Universitaetsklinikum Hamburg-Eppendorf, Hamburg, Germany

¹Correspondence: Experimentelle Ophthalmologie, Klinik und Poliklinik fuer Augenheilkunde, Universitaetsklinikum Hamburg-Eppendorf, Martinistraße 52, Hamburg 20246, Germany. E-mail: o.strauss{at}uke.uni-hamburg.de

SPECIFIC AIMS

Mutations in the VMD2 gene cause Best’s vitelliform macular degeneration. The gene product of VMD2, bestrophin-1, has been described as a Ca²⁺-dependent Cl channel. All presently analyzed mutants of bestrophin-1 showed a loss of Cl channel function. This would explain the reduction of the light-peak amplitude in the patient’s electro-oculogram (EOG) resulting from reduced activation of Cl channels in the basolateral membrane of the retinal pigment epithelium.

However, in several recently published papers patients with mutations in the VMD2 gene show a great heterogeneity in the clinical picture, such as macular degeneration accompanied by a normal EOG. This raises the question of additional functional aspects of bestrophin-1.

PRINCIPAL FINDINGS

1. L-type Ca²⁺ channels participate in the activation of the light-peak
In previous studies, we showed that inositol-1,4,5-trisphosphate-dependent activation of Ca²⁺-dependent Cl channels involves an influx of extracellular Ca²⁺ into the cell through L-type Ca²⁺ channels. Since the light-peak results from activation of Cl channels in the basolateral membrane of the RPE, the contribution of L-type channels in the increase of the light-peak was investigated. Systemic application of the dihydropyridine L-type channel blocker nimodipine resulted in significant reduction of the light-peak amplitude in the DC-electroretinogram of the rat. Nimodipine did not alter heart rate or blood pressure of the mice. Unchanged a- and b-waves indicate that altered photoreceptor activity and altered activity of retinal neurons do not account for the observed changes in the DC-electroretinogram.

2. Cells of the RPE-J cell line express L-type Ca²⁺ channels
To study possible influences of bestrophin-1 on L-type Ca²⁺ channels the RPE-J cell line was chosen as expression system. This rat-derived RPE cell line has a nondetectable endogenous expression of bestrophin-1. To analyze Ca²⁺ channel currents, perforated patch-clamp recordings under intracellular and extracellular K⁺-free conditions were performed using a pipette solution with low intracellular Ca²⁺ concentration to avoid activation of Ca²⁺-dependent Cl channels. In the presence of 10 mM Ba²⁺ in the bath solution as charge carrier, the cells responded to depolarization from a holding potential of –70 mV to values more positive than –30 mV with activation of fast activating and inactivating inward currents. The currents were reduced by 60% in the presence of 10 µM nifedipine, showed a half maximal activation at –14 mV and a fast activation with a time-to-peak value of 5 ms. Western blot analysis revealed the expression of L-type channel Ca_V1.3 subunits and not Ca_V1.2 subunits. Thus, cells of the RPE-J cell line express L-type Ca²⁺ channels that have also been found in fresh or cultured RPE cells from various species.

3. Expression of wild-type bestrophin changed time- and voltage-dependent activation of L-type channels in RPE cells
Transfection of RPE-J cells with eGFP alone did not affect L-type channel characteristics. Cotransfection with bestrophin-1 and eGFP resulted in different L-type Ca²⁺ channel properties. Bestrophin-1 was detected in the cell membrane of transfected cells. With bestrophin-1, L-type channel currents showed faster time-dependent activation and a shift of the voltage-dependent activation toward more negative potentials (Fig. 1 ). Analysis of the time-to-peak values over the whole voltage-range at which L-type channels are active indicated that the variations in the activation kinetics were not due to the shift in the voltage-dependent activation. This indicates that in the presence of wild-type bestrophin-1 L-type Ca²⁺ channels show distinct alterations in their voltage- and time-dependent activation. The shift in the voltage-dependent activation increases the number of active channels near the resting potential of RPE cells. The faster activation would facilitate Ca²⁺ mobilization as a second-messenger to regulate distinctive changes in cell function.

View larger version (37K): [in this window] [in a new window]   Figure 1. Effect of wild-type bestrophin-1 transfection. A) RPE-J cells were cotransfected with vectors encoding GFP and one of the following: empty vector (sham), bestrophin-1 (WT), bestrophin-W93C (W93C), or bestrophin R218C (R218C). After 24 h, cells were surface biotinylated. After lysis, bestrophin or GFP were immunoprecipitated as indicated and blotted for biotin using streptavidin, for bestrophin using an anti-Best-1 monoclonal antibody, or for GFP using an anti-GFP monoclonal antibody. Note that WT, W93C, and R218C forms of bestrophin were biotinylated and present on the cell surface, in contrast to GFP, an intracellular protein. B) Currents activated by the electrical stimulation shown in panel A; cell is transfected with bestrophin-1 and eGFP as reporter. C) Direct comparison of kinetics of L-type currents between a cell transfected with eGFP alone and a cell cotransfected with eGFP + bestrophin-1. Currents were normalized to the maximal current amplitude to allow a direct comparison. D) Fluorescence microscopy: RPE-J cell transfected with eGFP-bestrophin-1 fusion construct showing bestrophin-1 localization in the cell membrane. E) Currents activated by the electrical stimulation shown in panel A; cell transfected with the eGFP-bestrophin-1 fusion construct. Recording derived from a cell showing a bestrophin-1 localization in the cell membrane.

4. Mutant bestrophin-1 led to different changes in L-type Ca²⁺ channel activity
To verify the effects of bestrophin-1 on L-type Ca²⁺ channels and to get first insights into the pathophysiology of Best’s disease, the mutants W93C and R218C were investigated. Both were described as frequently observed mutants. Transfection with W93C showed a shift in the voltage-dependent activation of L-type channels comparable to that observed with wild-type bestrophin-1. However, cells expressing this mutant exhibited L-type channel currents with different kinetic properties than cells with wild-type bestrophin-1. Here, L-type channel currents displayed significantly prolonged time-dependent activation and inactivation. The time-to-peak value at the maximal current was 3-fold larger and the inactivation time constant was 5-fold larger than in cells transfected with wild-type bestrophin-1. Transfections using the mutant R218C shifted the voltage-dependent activation to more negative values as compared with wild-type bestrophin-1. In the presence of R218C bestrophin-1, the time-to-peak values of L-type currents were comparable to that observed with wild-type bestrophin-1. However, in the presence of this mutant the L-type channels showed a much faster inactivation. Thus mutant bestrophin-1 changed properties of L-type Ca²⁺ channels. The way the mutant affected L-type channel activity was dependent on the type of mutation. This further supports the conclusion that bestrophin-1 specifically modulates activity of L-type channels in the RPE.

CONCLUSIONS AND SIGNIFICANCE

The inhibitory effects of the L-type channel blocker nimodipine on the light-peak in the DC-electroretinogram of the rat indicates that these Ca²⁺ channels participate in the generation of this signal that derives from changes in the membrane conductance of the RPE. The expression of L-type channels in the RPE has been shown in several studies using freshly isolated or cultured RPE cells from various species. However, the nimodipine effects on the light-peak demonstrate for the first time that these ion channels play a functional role in the RPE in the intact eye.

RPE-J cells transfected with bestrophin-1 showed L-type channel currents that activate at more negative potentials and much faster. Mutant bestrophin-1 also influenced L-type channel activity but with different features. Thus, bestrophin-1 appeared as a modulator of L-type channels. Most important is a shift of the voltage dependence to more negative values, closer to the resting potential of RPE cells. With this shift these voltage-dependent channels can contribute to changes in intracellular free Ca²⁺ in epithelial cells, which rarely show prominent changes in their membrane potential. These effects of bestrophin-1 might help to understand differences of Ca²⁺ channel properties in heterologeous expression systems and in native cells. L-type channels composed of Ca_V1.3 subunits are particularly known to display a wide range in voltage dependence.

The mutant bestrophin-1 mainly changed the efficiency with which L-type channels can conduct Ca²⁺ into the cell. The changes observed in the expression system correspond with modulations of the light-peak in a previously published rat model for Best’s disease.

So far, bestrophins have been convincingly described as Ca²⁺-depdendent Cl channels. The function of bestrophin-1 as a Cl channel is not mutually exclusive. It is likely that bestrophin-1 combines the function of Cl channel and Ca²⁺ channel regulator and brings both ion channel types together into one signaling complex. This would provide an elegant feedback mechanism between Ca²⁺ homeostasis and Cl conductance of the cell membrane. It would open new ways to understand the heterogeneity in the clinical picture of patients carrying VMD2 mutations.

View larger version (22K): [in this window] [in a new window]   Figure 2. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4495fje;

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