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Abstract

We have characterised the spectroscopic properties of the metallochromic dye dichlorophosphonazo III and describe its use for the determination of changes of Mg²⁺ concentration in the micromolar range. Using a previously described reconstitution procedure, we incorporated the cGMP-gated channel from bovine rod photoreceptors into magnesium-containing liposomes and used the dye to monitor cGMP-activated Mg²⁺-efflux. The Km and cooperativity of the cGMP-dependence were identical regardless of whether Mg²⁺ or Ca²⁺ was the transported ion, however, the vmax for Ca²⁺ was more than 2-fold higher than that for Mg²⁺. We thereby determined a channel selectivity (Ca²⁺:Mg²⁺) of 1.0:0.44 in the presence of symmetrical (30 mM) K+. We also describe conditions where Mg²⁺ or Ca²⁺ effluxes can be selectively monitored in the presence of each other. This allowed the demonstration that magnesium ions can flow through the cGMP-gated channel even in the presence of an identically directed calcium gradient. Together these results indicate that magnesium ions may enter the photoreceptor rod outer segment cytosol through the cGMP-gated channel under dark conditions, thereby alluding to the existence of an as yet unknown Mg²⁺-extrusion mechanism, distinct from the Na⁺/Ca²⁺-exchanger, in these cells.