Localization of type I inositol 1,4,5-triphosphate receptor in the outer segments of mammalian cones

Abstract

Calcium enters the outer segment of a vertebrate photoreceptor through a cGMP-gated channel and is extruded via a Na/Ca, K exchanger. We have identified another element in mammalian cones that might help to control cytoplasmic calcium. Reverse transcription-PCR performed on isolated photoreceptors identified mRNA for the SII- splice variant of the type I receptor for inositol 1,4,5-triphosphate (IP3), and Western blots showed that the protein also is expressed in outer segments. Immunocytochemistry showed type I IP3 receptor to be abundant in red-sensitive and green-sensitive cones of the trichromatic monkey retina, but it was negative or weakly expressed in blue-sensitive cones and rods. Similarly, the green-sensitive cones expressed the receptor in dichromatic retina (cat, rabbit, and rat), but the blue-sensitive cones did not. Immunostain was localized to disk and plasma membranes on the cytoplasmic face. To restore sensitivity after a light flash, cytoplasmic cGMP must rise to its basal level, and this requires cytoplasmic calcium to fall. Cessation of calcium release via the IP3 receptor might accelerate this fall and thus explain why the cone recovers much faster than the rod. Furthermore, because its own activity of the IP3 receptor depends partly on cytoplasmic calcium, the receptor might control the set point of cytoplasmic calcium and thus affect cone sensitivity.

Fig. 1.

Mammalian retina expresses type I IP₃receptor. A, Diagram of type I IP₃ receptor mRNA and location of PCR primers (arrows).B, RT-PCR of the SII-containing region of type I IP₃ receptor on rat whole retina (WR) and isolated photoreceptors (PR). L, DNA molecular weight ladder. C, Differential interference image of two groups of isolated rat photoreceptors used for RT-PCR. The outer segment (OS) and inner segment (IS) are indicated. D, Western blots of protein extracts from rat and cat probed with C-terminus antibody against type I IP₃ receptor. For rat, two protein concentrations (7.5 and 15 μg) from whole retina were loaded. For cat, 15 μg was loaded.OS, Outer segments; WR, whole retina.Arrows point to type I IP₃ receptor-positive band at the predicted molecular weight.

Fig. 2.

Type I IP₃ receptor is localized to cone outer segments. A, Frozen radial sections immunostained for type I IP₃ receptor with C-terminus antibody. All species show strong staining in cone outer segments. In rabbit, the cells located in the outer layer of the ONL also stain strongly; their location and distribution suggest that these are cone somas. GCL, Ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer;IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments. B, Left, Rat section immunostained with antibody “M” directed against an internal domain of type I IP₃ receptor. Cone outer segments are stained distinctly. B, Right, Rat section stained with the preimmune serum is devoid of stain.

Fig. 3.

S cones do not stain for type I IP₃ receptor. A, B, Monkey retina stained with antibodies against type I IP₃ receptor (A; visualized with DAB reaction product) and blue-sensitive opsin (B; visualized with Cy3).Arrows point to S cone outer segments that are negative for type I IP₃ receptor but are positive for blue-sensitive opsin. C, D, Monkey retina stained with antibodies against type I IP₃ receptor (C; FITC) and red/green opsin (D; rhodamine). All cone outer segments stained for type I IP₃ receptor are also positive for red- and green-sensitive opsin. E–G, Cat retina stained with antibodies against type I IP₃ receptor (E; DAB) and blue-sensitive opsin (F; Cy3). Dotted outlines in E designate the location of the blue cone outer segments. G, Simultaneous visualization of both stainings: cone outer segments stained for the blue opsin do not stain for type I IP₃receptor.

Fig. 4.

Stain for type I IP₃ receptor in cones is stronger than in rods, and it is also present in connecting cilia (monkey). A, Light micrograph of a 1 μm Epon section; cone outer segments (cone OS) are much stronger than rod outer segments. Arrows indicate stained connecting cilia in rods; the arrowhead indicates stained connecting cilium in a cone. B, Electron micrograph of a rod connecting cilium (c). Arrowsindicate the staining (gold deposits) along the tubular structures of the connecting cilium. IS, Inner segment;OS, outer segment.

Fig. 5.

Type I IP₃ receptor is localized to cone and rod disk membranes (monkey). A, In the cone outer segment the immunodeposits are dense. In a fixed tissue the hypertonic condition often causes the disk membrane to collapse, which leads to a narrow intradisk lumen and wide interdisk space (or cytoplasmic space). Almost all of the immunodeposits are in the cytoplasmic space. B, In the rod outer segments the immunodeposits are scattered.

Fig. 6.

Staining for type I IP₃ receptor is present on the cytoplasmic face of the plasma membrane.A, Monkey cone outer segment. Arrowheadsindicate disk lumen (dl) and plasma membrane (pm). Arrows indicate that the staining is associated at the cytoplasmic side of disk and plasma membranes. B, In rat it is difficult to discriminate cones from rods, but because rods are 100-fold more abundant and most neighboring outer segments appear similar to this one, we think that it is a rod outer segment. Arrows indicate that the staining is associated at the cytoplasmic side of disk membranes.

Fig. 7.

How the IP₃ receptor might contribute to response recovery and adaptation. Solid arrows mark the phototransduction cascade leading from light (hυ) to the successive activation of opsin (Rh*), transducin (T*), phosphodiesterase (PDE*), and the hydrolysis of cGMP. Cation channels gated by cGMP close, thereby reducing Ca²⁺ influx, but Ca²⁺extrusion continues, so cytoplasmic Ca²⁺ falls. Low cytoplasmic Ca²⁺ affects several processes that terminate the light response and contribute to the response recovery (dotted arrows): opsin is phosphorylated, guanylyl cyclase (GC) is activated to synthesize cGMP, and channel affinity for cGMP is increased by binding Ca²⁺/calmodulin (CaM). The IP₃ receptor (IP3R) on the disk and plasma membranes would accelerate changes in Ca²⁺_i (dashed arrows). When Ca²⁺ and cGMP fall, phospholipase C (PLC) is suppressed, reducing IP₃. Because both IP₃ and Ca²⁺ regulate the IP₃ receptor, their fall reduces Ca²⁺mobilization from the disks and extracellular space. This positive feedback loop via the IP₃ receptor should accelerate the fall of Ca²⁺ after a light stimulus and its rise after a dark stimulus.