In sensory neurons, successful maturation of signaling molecules and regulation of Ca²⁺ are essential for cell function and survival. A multifunctional role for calnexin has been demonstrated as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca²⁺ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca²⁺ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration; this degeneration is enhanced by light, suggesting that calnexin's function as a Ca²⁺ buffer is important for photoreceptor cell survival. These results illustrate a critical role for calnexin in Rh1 maturation and Ca²⁺ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration (Rosenbaum, 2006).

G protein-coupled receptors are synthesized on membrane-bound ribosomes and undergo translocation, modification, folding, oligomeric assembly, and quality control in the endoplasmic reticulum (ER). To deal with these complex and error-prone processes, the ER has evolved a system centered around the calnexin family of molecular chaperones; calnexins promotes the proper folding and assembly of newly synthesized glycoproteins. Calnexin is a type I transmembrane protein that, like its soluble ER homolog calreticulin, interacts with the monoglucosylated glycan (Glc₁Man_7-9GlcNAc₂) present on folding intermediates of glycoproteins. Nascent glycoproteins associate with calnexin or calreticulin via a cycle of binding and release (Ellgaard, 2003, Molinar, 2004, Schrag, 2001; Ware, 1995). This cycle is key for ER quality control, since it inhibits aggregation, prevents premature exit from the ER, and exposes glycoproteins to accessory enzymes and folding factors (Ellgaard, 2003). Despite considerable understanding of the calnexin/calreticulin cycle, little is known about the requirement for calnexin in protein processing in vivo (Rosenbaum, 2006).

Rhodopsin is the prototypical member of the large G protein-coupled receptor family. As in vertebrates, Drosophila rhodopsin (Rh1) initiates the phototransduction cascade by interacting with a heterotrimeric G protein, which then activates a distinct effector enzyme, namely phospholipase C (PLC-β). Activation of PLC leads to the opening of the cation-selective TRP and TRPL channels, resulting in a dramatic rise in intracellular Ca²⁺ (Rosenbaum, 2006 and references therein).

To become functionally active, newly synthesized rhodospin must be precisely folded and successfully navigate the secretory pathway to the phototransducing compartment of the photoreceptor cells, the rhabdomeres. Rhabdomeres consist of numerous tightly packed microvilli containing the phototransduction machinery. The mechanisms that regulate the folding and transport of rhodopsin are essential for photoreceptor cell function and survival, since defects in rhodopsin maturation led to retinal degeneration in both Drosophila and vertebrates. Protein maturation defects characterized in blinding diseases have broad implications, because protein misfolding and aggregation are characteristic of a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. This study investigates whether calnexin functions as a chaperone for Rh1 and whether mutations in calnexin lead to neurodegeneration (Rosenbaum, 2006).

In addition to its role as a molecular chaperone, calnexin is thought to bind Ca²⁺ at two distinct sites. The first resides in the long N-terminal domain localized to the lumen of the ER. The crystal structure of vertebrate calnexin shows that this luminal domain consists of two distinct regions: a compact, globular domain and a proline-rich arm called the P domain. The globular domain is thought to bind a single Ca²⁺ ion and is also involved in glucose binding (lectin domain) (Schrag, 2001). Although less well defined, several lines of evidence suggest that calnexin may harbor a second Ca²⁺-binding domain within the highly charged C-terminal cytosolic tail (C domain). Interestingly, this cytosolic domain displays structural similarity to calreticulin's luminal C domain, but is positioned on the opposite, cytosolic side of the ER membrane (Tjoelker, 1994). Calreticulin's C domain displays low-affinity and high-capacity Ca²⁺ binding and is thought to buffer luminal Ca²⁺ (Baksh, 1991). While the in vivo role of calnexin's C domain is unknown, these Ca²⁺-binding properties would make it ideal for buffering high concentrations of cytosolic Ca²⁺ (Rosenbaum, 2006).

Precise spatial and temporal control over Ca²⁺ levels is essential for phototransduction in both vertebrates and invertebrates. Furthermore, prolonged elevation of cytosolic Ca²⁺ can be toxic, leading to cell death. In Drosophila, the light-sensitive TRP and TRPL channels mediate a massive Ca²⁺ influx into the rhabdomeres that is essential for amplification, rapid-response kinetics, and light adaptation. Ca²⁺ can rise to about 1 mM in the rhabdomeres and is removed from the rhabdomeres by a combination of the Na⁺/Ca²⁺ exchanger (CalX) and diffusion into the cell body, where Ca²⁺ rises to about 10 μM. The mechanisms controlling Ca²⁺ in the cell body are poorly understood but presumably include sequestration by the sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) and buffering by cytosolic Ca²⁺-binding proteins. This study explores the hypothesis that calnexin serves as a buffer for cytosolic Ca²⁺ following the acute Ca²⁺ rise during phototransduction and that it may thereby help to prevent Ca²⁺ toxicity and promote photoreceptor cell survival (Rosenbaum, 2006).

This report demonstrates a multifunctional role for calnexin as both a critical molecular chaperone for Rh1 biosynthesis and a regulator of cytosolic Ca²⁺. Mutations in calnexin cause retinal degeneration that is enhanced by light, suggesting that calnexin's role in Ca²⁺ removal may be important for photoreceptor cell survival. These results provide genetic evidence that failure in Rh1 maturation and Ca²⁺ overload, resulting from defects in calnexin, are responsible for retinal degeneration (Rosenbaum, 2006).

Thus Cnx plays a multifunctional role, serving as a chaperone for Rh1 and a regulator of Ca²⁺ during phototransduction. Furthermore, genetic evidence shows that failure in Rh1 maturation and Ca²⁺ overload, resulting from defects in cnx, are responsible for retinal degeneration (Rosenbaum, 2006).

Calnexin serves a key role in glycoprotein folding and quality control in the ER and is thought to interact with a wide variety of newly synthesized proteins. Consistent with calnexin's broad substrate specificity, it is expressed at all stages during development and in the adult. However, cnx mutants are homozygous viable and fertile, indicating that all proteins required for survival and reproduction fold sufficiently in the absence of Cnx. Cnx is, however, essential in the eye. Loss-of-function mutations in cnx lead to defects in Rh1 maturation and cause age-dependent retinal degeneration. Although Cnx is essential for Rh1 biosynthesis, it is not required for the expression or function of other photoreceptor cell proteins. This indicates that even within the eye, Cnx may play a unique role. There are two possible explanations for the specific requirement for Cnx by Rh1: (1) chaperones with redundant functions may compensate for the loss of Cnx; (2) Cnx may serve as a chaperone that is exclusively dedicated to Rh1 (Rosenbaum, 2006).

With the exception of Rh1, photoreceptor cell proteins manage to fold normally in cells lacking calnexin. This could be due to redundancy between folding factors in the ER. For example, calreticulin (located on 3R, 85E) as well as two additional calnexin genes (cnx11 and cnx14) are present in Drosophila. These chaperones may compensate for the lack of calnexin function in all cases except during Rh1 biosynthesis. An alternative conclusion is that Cnx may be exclusively devoted to Rh1. Consistent with this scenario, another chaperone, NinaA, is also expressed in all eight photoreceptor cells yet is specifically dedicated to Rh1 biosynthesis in the R1-6 photoreceptor cells. In addition, a cyclophilin-like protein expressed in the mammalian retina, RanBP2, acts as a specific chaperone for red/green opsin. Given the importance and abundance of rhodopsin, it is plausible that Cnx and NinaA are part of a system of chaperones that is dedicated to its folding and quality control (Rosenbaum, 2006).

Consistent with results presented in this study, calnexin-deficient mice have been generated that are homozygous viable. Half die within the first 2 days, and those that survive are smaller than their littermates, develop severe motor disorders, and display a dramatic loss of large myelinated nerve fibers (Denzel, 2002). Mice contain a single calnexin gene, eliminating the possibility of redundancy between calnexins. However, it is possible that calreticulin is able to partially compensate for the loss of calnexin in utero and in adults, but is unable to compensate for all calnexin functions (Rosenbaum, 2006).

Photoreceptor cells in the cnx mutants display elevated and sustained cytosolic Ca²⁺ levels following light stimulation, consistent with a role for Cnx in buffering Ca²⁺. Calnexin's cytosolic domain displays structural similarity to calreticulin's highly charged C domain, but is positioned on the opposite side of the ER membrane (Tjoelker, 1994). Calreticulin's C domain displays low-affinity, high-capacity Ca²⁺-binding properties and plays a major role in Ca²⁺ modulation in the lumen of the ER (Baksh, 1991). Given their structural similarity, it is proposed that calnexin's C domain serves a similar function in modulating Ca²⁺, but in the cyotosol. These low-affinity and high-capacity Ca²⁺-binding properties would make calnexin's cytosolic domain ideal for buffering high levels of Ca²⁺ in the photoreceptors. It is proposed that Cnx serves to buffer Ca²⁺, which diffuses into the cell body after entering via the light-sensitive TRP and TRPL channels (Rosenbaum, 2006).

The Drosophila compound eye has emerged as an important model for unraveling the mechanisms of retinal degeneration and phototransduction. Retinal degeneration can be triggered by mutations in almost every protein that functions in phototransduction. Although the mechanisms for each are not well understood, these mutations can be divided into at least two distinct classes. One involves protein maturation defects, most commonly in rhodopsin, and the other involves a combination of unregulated activities of the phototransduction cascade and/or Ca²⁺ toxicity. Retinal degeneration caused by defects in rhodopsin folding does not require light activation of phototransduction and is therefore light independent. However, retinal degenerations stemming from unregulated phototransduction or Ca²⁺ toxicity are dependent on light stimulation of the cascade and opening of the light-sensitive TRP and TRPL channels. Genetic analysis has revealed a number of mutants that fall into one class or the other, but very few that exhibit both properties (Georgiev, 2005). The cnx mutants clearly display defects in Rh1 maturation, and yet they also undergo a light-enhanced retinal degeneration. This indicates that defects in Rh1 maturation are not solely responsible for the retinal degeneration. The results, indicating impaired Ca²⁺ buffering in the cnx mutants, along with the ability of the norpA mutation to partially prevent degeneration in cnx, suggests that Ca²⁺ toxicity also contributes to the retinal degeneration. Therefore, it is proposed that the cnx mutant displays characteristics of two distinct classes of retinal degeneration; one involving defects in Rh1 maturation, and the other involving Ca²⁺ toxicity (Rosenbaum, 2006).

These results provide genetic evidence that failure in Rh1 maturation and Ca²⁺ overload, resulting from defects in Cnx, are responsible for retinal degeneration. Because calnexin has been localized to the ER of photoreceptor cells in mice, it may play a protein-folding role in the mammalian retina as well as in Drosophila (Frederick, 2001). Furthermore, because Drosophila cnx displays 49% amino acid identity with human calnexin, mutations identified in Drosophila calnexin may be clinically relevant to hereditary human retinal degeneration diseases (Rosenbaum, 2006).