ATP-dependent calcium uptake activity associated with a disk membrane fraction isolated from bovine retinal rod outer segments

Abstract

Ca2+ sequestration and release from disks of rod outer segments may play a critical role in visual transduction. An ATP-dependent Ca2+ uptake activity has been identified in association with purified disks of bovine rod outer segments. A crude preparation of osmotically active disks was obtained from rod outer segments by hypoosmotic shock and subsequent flotation on a 5% Ficoll 400 solution. These "crude" disks were further purified by separation into two distinct components by centrifugation in a linear Ficoll gradient. Disks comprised the major component; at least 60% of the protein was rhodopsin. This fraction also contained a Ca2+ uptake activity stimulated approximately 4-fold by ATP. The initial rate was approximately 0.21 nmol of Ca2+ (mg of protein)-1 min-1. Most of the ATP-dependent accumulation of 45Ca2+ was released by the calcium ionophore A23187. The uptake activity was sensitive to vanadate (Ki approximately 20 microM) and insensitive to the mitochondrial Ca2+ uptake inhibitor ruthenium red (10 microM). The ATP-dependent Ca2+ uptake exhibited Michaelis-Menten activation kinetics with respect to [Ca2+] (Km approximately 6 microM). The osmotic properties of the sealed disk membranes were exploited to determine whether the association of Ca2+ transport activity with the disks was merely coincidental. The sedimentation properties of these disks, upon centrifugation on a second Ficoll linear density gradient, varied with the osmolarity of the gradient solution. In several separate gradient solutions of differing osmotic and ionic strengths, the Ca2+ uptake activity always comigrated with the disks. These results indicate that the ATP-dependent Ca2+ uptake activity was physically associated with sealed native disk membranes. The characteristics of the Ca2+ uptake activity suggest that it may play a major role in the regulation of cytosolic Ca2+ levels in rod cells in vivo.

Fibroblast growth factor phosphorylation and receptors in rod outer segments.

“…The second one, according to Uhl et al (1987) (method B, see Materials and methods) is considered to yield predominantly intact ROS. To assess the role of ROS plasma membrane in the following experiments, we have also enriched membrane preparations for the disc in accordance with Puckett et al (1985). The Coommassie Blue/silver stained PAGE profile of the ROS and disc preparations is shown in Figure 4.…”

Evidence for two functionally different membrane fractions in bovine retinal rod outer segments.

“…In contrast to this conclusion, Puckett & Goldin (1986) report cyclic GMPdependent Ca2+ release from 'purified disc preparations'. The purification procedure employs two ficoll centrifugation steps, using up to 20 % (w/v) concentrations of ficoll (Puckett, Aronson & Goldin, 1985). It is possible, however, that separation of disc and plasma membranes in such high ficoll concentrations is hampered by interaction of ficoll with outer segment membrane vesicles (Bauer & Mavrommati, 1986).…”

Serca isoform expression in the mammalian retina

“…Furthermore, following exposure to light, [Ca 2+ ] OS falls to lower baseline levels than in rods (Fain et al, 2001). Although some early biochemical studies suggested that ATP-dependent Ca 2+ pumps are responsible for Ca 2+ sequestration in rod OSs (Mason et al, 1974;Kaupp et al, 1981;Schnetkamp and Kaupp, 1985;Puckett et al, 1985;Pepe et al, 2000) and that this effect is proton-dependent (Kaupp, 1981;Schnetkamp and Kaupp, 1983; as has been shown for SERCA-mediated Ca 2+ transport (Naderali et al, 1997), there is at present no physiological evidence that ATP-driven Ca 2+ pumps contribute to Ca 2+ extrusion within the cone outer segment; the Na + -K + , Ca 2+ exchangers are thought to act as the sole Ca 2+ extruders at the cone OS plasma membrane (Nakatani and Yau, 1985;Prinsen et al, 2000;reviewed in Fain et al 2001). …”