The effect of light on outer segment calcium in salamander rods

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Abstract
Calcium acts as a second messenger in vertebrate rods, regulating the recovery phase of the light response and modulating sensitivity during light-adaptation. Since light not only decreases the outer segment calcium concentration ([Ca2+]i) by closing cyclic nucleotide-gated channels but can also increase [Ca2+]i by releasing Ca2+ from buffer sites or intracellular stores, we examined in detail the effect of light and circulating current on [Ca2+]i by making simultaneous measurements of suction pipette current and [Ca2+]i from isolated rods of the salamander Ambystoma tigrinum after incorporation of the fluorescent dye fluo-5F. When the release of Ca2+ is measured in 0 Ca2+−0 Na+ solution, minimising fluxes of Ca2+ across the plasma membrane, it is substantial only for light bright enough to bleach a significant fraction of the photopigment and is restricted to the part of the outer segment in which the bleach occurred. It is unlikely, therefore, to make a large contribution to [Ca2+]i for most of the physiological operating range of the rod. Nevertheless, since release is half-maximal for a bleach of less than 10 %, it cannot be produced by a simple mechanism such as a change in the affinity of a binding site on rhodopsin itself but must instead require some more complex interaction. In Ringer solution, the Ca2+ in the light-releasable pool can be discharged merely by the decrease in [Ca2+]i that occurs as the outer segment channels close. In steady background light or after exposure to saturating illumination, the fraction of Ca2+ in the pool decreases essentially in proportion to [Ca2+]i as if Ca2+ were being removed from a buffer site within the cytoplasm. Furthermore, [Ca2+]i itself changes in proportion to the circulating current, with little evidence for a contribution from Ca2+ release or other mechanisms of Ca2+ homeostasis. This indicates that flux of Ca2+ across the plasma membrane is the major determinant of outer segment Ca2+ concentration within the rod's normal operating light intensity range. Once Ca2+ has been discharged from the releasable pool, it is restored following dim illumination apparently as the simple result of the subsequent restoration of dark [Ca2+]i and the rebinding of Ca2+ to its release site, but after brighter light perhaps also as a consequence of regeneration of the photopigment.