Spectrophotometric determination of photoreceptor cGMP-gated channel Mg2(+)-fluxes using dichlorophosphonazo III.

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.