Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurr ent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-5. When a dark-adapte d cone was exposed to the intense illumination of the laser, the circulatin g current was completely suppressed and fluo-3 fluorescence rapidly decline d. In the more numerous red-sensitive cones this light-induced decay in flu o-3 fluorescence was best fitted as the sum of two decaying exponentials wi th time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) an d unequal amplitudes: the faster component was 1.7-fold larger than the slo wer. In blue-sensitive cones, the decay in fluorescence was slower, with ti me constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitude s. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allo wed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after satura ting illumination (mean +/- SEM, n = 8). Photopigment bleaching by the lase r resulted in a considerable reduction in light sensitivity and a maintaine d decrease in outer segment Ca2+ concentration. When the photopigment was r egenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Reg eneration of the photopigment allowed repeated measurements of fluo-3 fluor escence to be made from a single red-sensitive cone during adaptation to st eady light over a range of intensities. These measurements demonstrated tha t the outer segment Ca2+ concentration declines in a graded manner during a daptation to background light, varying linearly with the magnitude of die c irculating current.