Bleached Pigment Produces a Maintained Decrease in Outer Segment Ca2+ in Salamander Rods

Abstract

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+ i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+ i estimates of 670 ± 250 nM in a dark-adapted rod and 30 ± 10 nM during response saturation after exposure to bright light (mean ± SD). The resting level of Ca2+ i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 ± 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+ i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+ i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

The Y99C Mutation in Guanylyl Cyclase-Activating Protein 1 Increases Intracellular Ca2+and Causes Photoreceptor Degeneration in Transgenic Mice

“…Photoreceptor membrane guanylyl cyclase (retGC1/2 or GC-E/F) (Garbers and Lowe, 1994;Garbers, 1999), regulated by GCAPs, subsequently restores the level of cGMP. Closure of the cGMP-gated channels in the light produces a decrease in outer segment Ca 2ϩ concentration (GrayKeller and Detwiler, 1994;Sampath et al, 1998Sampath et al, , 1999Woodruff et al, 2002), causing Ca 2ϩ to dissociate from the GCAPs, and that stimulates retGC to synthesize more cGMP and to reopen the cGMP-gated cation channels. This GCAP-dependent activation of the cyclase plays an important role both in shaping the photoreceptor light response and in light adaptation (Fain et al, 2001;Mendez et al, 2001;Burns et al, 2002).…”

Amino Acid Residues of S-modulin Responsible for Interaction with Rhodopsin Kinase

“…These modulations are attained by changes in the cytoplasmic Ca 2ϩ concentration (3,4). In the dark, the cytoplasmic Ca 2ϩ concentration in a rod is in a range of several hundred nanomolar (5,6). Under this condition, the photoresponse saturates at low levels of light and the light sensitivity is high.…”

“…Under this condition, the photoresponse saturates at low levels of light and the light sensitivity is high. In the light, the Ca 2ϩ concentration decreases to ϳ0 -30 nM under a very bright light (5,6). Under light-adapted conditions, the operating range shifts to a brighter region and the light-sensitivity decreases.…”

Dimerization of Guanylyl Cyclase-activating Protein and a Mechanism of Photoreceptor Guanylyl Cyclase Activation

“…Lowering of the intracellular Ca 2ϩ concentrations caused by illumination stimulates cGMP synthesis by guanylyl cyclase (retGC) 1 that contributes to recovery from photoexcitation and to light adaptation of photoreceptors (5,6). Recoverin-like Ca 2ϩ -binding proteins, GCAP-1 and GCAP-2, mediate Ca 2ϩ sensitivity of the cyclase in vertebrate retinas (7)(8)(9)(10) so that GCAPs activate the cyclase when the free concentrations of Ca 2ϩ decrease below 100 nM (a characteristic of light-adapted photoreceptors), but they do not stimulate the cyclase in the dark when the free Ca 2ϩ concentrations exceed 500 nM (6,11). GCAP-2 is highly abundant in mammalian rods, while GCAP-1 is highly expressed in cones (9,10,12,13).…”