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O. Strauss, A.D. Marmorstein, L.Y. Marmorstein, B. Bakall, J.B. Stanton, C. Wadelius, N.S. Peachey, R. Rosenthal; Bestrophin modulates activity of L–type Ca2+ channels in RPE cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1760.
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Purpose: Best vitelliform macular dystrophy is caused by mutations in the VMD2 gene encoding the protein bestrophin. The disease is characterized by a reduction of the light–peak in the EOG. Since the light–peak arises from an increase in basolateral Cl conductance, it has been hypothesized that bestrophin might be a Cl channel or a regulator of the Cl conductance. Our studies sought to identify the function of bestrophin in RPE cells. Methods: RPE–J cells were transiently transfected with bestrophin. Membrane currents of transfected cells were measured in the whole–cell configuration of the patch–clamp technique. Results:RPE cells with weak bestrophin expression showed bestrophin localisation in the plasma membrane. These cells showed no changes in the membrane conductance indicating expression of Cl channels. However, modulations of Ba2+ currents through L–type Ca2+ channels were detected. Bestrophin shifted the voltage–dependent activation towards more negative values (V1/2 non–transfected cells = –14.0 ± 0.7 mV, n = 19; versus transfected cells V1/2 = 22.3 ± 1.6 mV, n =12) and accelerated the activation kinetics (time–to–peak non–transfected 4.7 ± 0.6 ms, n = 16; versus transfected cells 1.3 ± 0.2; n = 9). Mutant bestrophin showed the same effect on the voltage–dependence but different modulations of the current kinetics. W93C bestrophin led to slower activation kinetics (time–to–peak 8.3 ± 3.4 ms, n = 5) and inactivation (wt–bestrophin inactivation time constant 7.8 ± 2.8 ms, n = 9; versus W93C 38.3 ± 12 ms, n = 3). R218C bestrophin led to faster inactivation kinetics (inactivation time constant 3.3 ± 1.1 ms; n = 6). Conclusions: Transfection of RPE cells revealed that bestrophin functions as a modulator of Ca2+ influx by influencing L–type Ca2+ channel activity. Reduced light–peak amplitude likely results from reduced capability to generate Ca2+ influx (too fast inactivation or to slow activation) and to activate Ca2+–dependent Cl channels.
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