Simultaneous measurement of current and calcium in the ultraviolet-sensitive cones of zebrafish

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In rods and visible cone photoreceptors, multiple measurements cannot be made of intracellular Ca2+ concentration from the same cell using fluorescent dyes, because a single exposure of the measuring light bleaches too large a fraction of the rod or cone photopigment. We have therefore identified and characterized UV-sensitive cones of the zebrafish, whose wavelength of maximum sensitivity is at 360 nm which is far enough from the wavelength of our measuring light (514.5 nm) so that it has been possible to make multiple determinations of photocurrent and Ca2+ concentration from the same cells. We show that for a limited number of measurements, for which the bleaching of the cone photopigment is too small to affect flash kinetics, the outer segment Ca2+ concentration closely follows the wave form of the flash response convolved with the dominant time constant for Ca2+ removal by Na+–Ca2+–K+ exchange. For a larger number of measurements, significant acceleration of the response kinetics by pigment bleaching inevitably occurs, but the Ca2+ concentration nevertheless rises and falls in approximate agreement with the flash wave form. During exposure to steady background light, the Ca2+ concentration falls in proportion to the steady-state current for dim backgrounds at all times and for bright backgrounds at steady state. At early times following the onset of bright backgrounds, however, the Ca2+ concentration is markedly higher than expected from the current of the cone. We show this to be the result of light-dependent Ca2+ release by bright background light, which can be abolished by pre-exposure of the cone to the membrane-permeant acetoxymethyl ester of the Ca2+ chelator BAPTA. Our results therefore demonstrate that the cone outer segment Ca2+ concentration is predominantly a function of the rate of influx and efflux of Ca2+ across the plasma membrane, but that a release of Ca2+ in bright light most probably from buffer sites within the cell can transiently elevate the Ca2+ concentration above the level expected from the open probability of the light-dependent channels.

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