Light-dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors

Abstract

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent 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-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 ± 2.4 and 640 ± 55 ms (mean ± SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 ± 30 and 1,400 ± 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed 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 saturating illumination (mean ± SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Calcium regulation in photoreceptors

“…In addition to more Ca 2+ entering into the cone OS relative to the rod OS, cone OSs are also more efficient at Ca 2+ extrusion. Extrusion of Ca 2+ is 4-8 times faster in cones compared to rods (7)(8)(9). This is consistent with a much faster and efficient light adaptation in cones compared to rods.…”

“…In the dark, the cytoplasmic Ca 2+ concentration in both rods and cones is ~ 300-500 nM (8,(14)(15). When CNG channels close following illumination, the influx of Ca 2+ stops but the extrusion continues, leading to a decrease in [Ca 2+ ]i the magnitude of which depends on the fraction of channels closed.…”

“…When CNG channels close following illumination, the influx of Ca 2+ stops but the extrusion continues, leading to a decrease in [Ca 2+ ]i the magnitude of which depends on the fraction of channels closed. In saturating light [Ca 2+ ]i can decrease to 20-50 nM in rods and < 10 nM in cones (8,(14)(15)20). Photoreceptor OSs also contain significant internal Ca 2+ stores whose function in the phototransduction and/or light adaptation is as yet unknown.…”

Serca isoform expression in the mammalian retina

“…This issue might be resolved in Ca 2+ imaging experiments from isolated mouse cones (e.g. ; Sampath et al 1999). However, Wang et al (1999) recently provided data that support indirectly localization of an integral endoplasmic reticulum protein to plasma membrane of cone OSs.…”

Ca2+ and Mg2+ Binding Properties of GCAP-1

“…We found the following. (i) At the physiological concentrations of free Mg 2ϩ and Ca 2ϩ that exist in the vertebrate photoreceptors in the light (3,23,24), three EF-hands in GCAP-1 are predominantly occupied with Mg 2ϩ ions; hence the physiological RetGC-activating form of GCAP-1 is triple Mg 2ϩ -bound form.…”