Alexander M. Dizhoor

Ectopic Expression of a Microbial-Type Rhodopsin Restores Visual Responses in Mice with Photoreceptor Degeneration

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.

The human photoreceptor membrane guanylyl cyclase, RetGC, is present in outer segments and is regulated by calcium and a soluble activator

A human photoreceptor membrane guanylyl cyclase, RetGC, was recently cloned and expressed, but its localization and manner of regulation were not defined. We report here that RetGC is detected primarily in outer segments of human photoreceptor cells. Recombinant RetGC can be stimulated by a soluble retinal-specific factor. Ca2+ interferes with stimulation of RetGC by this factor with a cooperativity coefficient of 1.7 and EC50 near 200 nM. The Ca2+ sensitivities of recombinant RetGC and of guanylyl cyclase activity from rod outer segment membranes are very similar. Our findings indicate that RetGC is a photoreceptor-specific guanylyl cyclase which is stimulated by a retinal-specific activator and inhibited by physiologically relevant concentrations of free Ca2+. The Ca2+ sensitivity of RetGC may be responsible for some of the previously reported effects of Ca2+ on light adaptation and recovery of the dark state.

Recoverin immunoreactivity in mammalian cone bipolar cells

Human, macaque monkey, and rat retinas were immunostained with a polyclonal antibody preparation against purified recoverin, a 23-kD calcium-binding protein isolated from bovine retina that localizes to rods and cones (Dizhoor et al., 1991). In addition to immunoreactive photoreceptors, we have identified subpopulations of recoverin-positive bipolar cells in all three species. Results from immunostaining with progressive dilutions of anti-recoverin and preadsorption of the antibody with a dilution series of purified recoverin showed that photoreceptors and bipolar cells had similar affinities for the antibody and suggested that the molecule recognized by the antibody in both cell types is recoverin. Immunoreactivity for recoverin and protein kinase C, a selective marker for all rod bipolar cells, was found in separate bipolar cell populations. Recoverin immunoreactivity is therefore a characteristic of certain cone bipolar cell types. In rat retina, anti-recoverin labeled two morphologically distinct subpopulations of cone bipolar cells whose axonal arbors stratified at different depths in the inner plexiform layer (IPL). The bipolar cells labeled with anti-recoverin did not correspond to those that were reactive for calbindin, another cone bipolar cell marker. Human and monkey retinas also had two populations of cone bipolar cells that were recoverin-positive. One population showed a distinct pattern of narrow bistratification at the outer border of the IPL and a regular mosaic arrangement of its axonal arbors, suggesting that the entire population of a single cone bipolar type was labeled. Cell density, dendritic morphology, and axonal-field size and stratification indicate that anti-recoverin selectively strains the flat midget (presumed OFF-center) cone bipolar cell type observed previously in Golgi preparations. By contrast the second bipolar cell population had axonal stratification in the inner half of the IPL and showed an unusual but consistent morphology and spatial distribution. Individual cells were intensely stained but were present at an extremely low density (approximately 2-5 cells/mm2). These cells had multibranched dendritic trees characteristic of the diffuse bipolar cell class, but very small axonal fields in the size range of the midget bipolar class. Neither of the two recoverin-positive bipolar cell types in monkey was labeled with anti-calbindin or anti-cholecystokinin. An antibody preparation against bovine pineal hydroxyindole-O-methyltransferase (HIOMT) labeled photoreceptors and bipolar cells that closely resembled the recoverin-positive bipolar cells in human and rat retinas.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors

The retinas photoreceptor cells adjust their sensitivity to allow photons to be transduced over a wide range of light intensities. One mechanism thought to participate in sensitivity adjustments is Ca2+ regulation of guanylate cyclase (GC) by guanylate cyclase-activating proteins (GCAPs). We evaluated the contribution of GCAPs to sensitivity regulation in rods by disrupting their expression in transgenic mice. The GC activity from GCAPs−/− retinas showed no Ca2+ dependence, indicating that Ca2+ regulation of GCs had indeed been abolished. Flash responses from dark-adapted GCAPs−/− rods were larger and slower than responses from wild-type rods. In addition, the incremental flash sensitivity of GCAPs−/− rods failed to be maintained at wild-type levels in bright steady light. GCAP2 expressed in GCAPs−/− rods restored maximal light-induced GC activity but did not restore normal flash response kinetics. We conclude that GCAPs strongly regulate GC activity in mouse rods, decreasing the flash sensitivity in darkness and increasing the incremental flash sensitivity in bright steady light, thereby extending the rods operating range.

Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases.

Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca2+ concentration (<100 nm) and inhibits them at high Ca2+ (>500 nm). The light-induced lowering of the Ca2+ level from ∼500 nm in the dark to ∼50 nm following illumination is known to play a key role in visual recovery and adaptation. We report here the three-dimensional structure of unmyristoylated GCAP-2 with three bound Ca2+ ions as determined by nuclear magnetic resonance spectroscopy of recombinant, isotopically labeled protein. GCAP-2 contains four EF-hand motifs arranged in a compact tandem array like that seen previously in recoverin. The root mean square deviation of the main chain atoms in the EF-hand regions is 2.2 Å in comparing the Ca2+-bound structures of GCAP-2 and recoverin. EF-1, as in recoverin, does not bind calcium because it contains a disabling Cys-Pro sequence. GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3. A prominent exposed patch of hydrophobic residues formed by EF-1 and EF-2 (Leu24, Trp27, Phe31, Phe45, Phe48, Phe49, Tyr81, Val82, Leu85, and Leu89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.

Constitutive Activation of Photoreceptor Guanylate Cyclase by Y99C Mutant of GCAP-1 POSSIBLE ROLE IN CAUSING HUMAN AUTOSOMAL DOMINANT CONE DEGENERATION

Photoreceptor membrane guanylate cyclases (RetGC) are regulated by calcium-binding proteins, GCAP-1 and GCAP-2. At Ca2+ concentrations below 100 nm, characteristic of light-adapted photoreceptors, guanylate cyclase-activating protein (GCAPs) activate RetGC, and at free Ca2+ concentrations above 500 nm, characteristic of dark-adapted photoreceptors, GCAPs inhibit RetGC. A mutation, Y99C, in human GCAP-1 was recently found to be linked to autosomal dominant cone dystrophy in a British family (Payne, A. M., Downes, S. M., Bessant, D. A. R., Taylor, R., Holder, G. E., Warren, M. J., Bird, A. C., and Bhattachraya, S. S. (1998) Hum. Mol. Genet. 7, 273–277). We produced recombinant Y99C GCAP-1 mutant and tested its ability to activate RetGC in vitro at various free Ca2+ concentrations. The Y99C mutation does not decrease the ability of GCAP-1 to activate RetGC. However, RetGC stimulated by the Y99C GCAP-1 remains active even at Ca2+ concentration above 1 μm. Hence, the cyclase becomes constitutively active within the whole physiologically relevant range of free Ca2+ concentrations. We have also found that the Y99C GCAP-1 can activate RetGC even in the presence of Ca2+-loaded nonmutant GCAPs. This is consistent with the fact that cone degeneration was dominant in human patients who carried such mutation (Payne, A. M., Downes, S. M., Bessant, D. A. R., Taylor, R., Holder, G. E., Warren, M. J., Bird, A. C., and Bhattachraya, S. S. (1998) Hum. Mol. Genet. 7, 273–277). A similar mutation, Y104C, in GCAP-2 results in a different phenotype. This mutation apparently does not affect Ca2+ sensitivity of GCAP-2. Instead, the Y104C GCAP-2 stimulates RetGC less efficiently than the wild-type GCAP-2. Our data indicate that cone degeneration associated with the Y99C mutation in GCAP-1 can be a result of constitutive activation of cGMP synthesis.

Calcium Binding, but Not a Calcium-Myristoyl Switch, Controls the Ability of Guanylyl Cyclase-activating Protein GCAP-2 to Regulate Photoreceptor Guanylyl Cyclase

Guanylyl cyclase-activating protein 2 (GCAP-2) is a recoverin-like calcium-binding protein that regulates photoreceptor guanylyl cyclase (RetGC) (Dizhoor, A. M., and Hurley, J. B. (1996)J. Biol. Chem. 271, 19346–19350). It was reported that myristoylation of a related protein, GCAP-1, was critical for its affinity for RetGC (Frins, S., Bonigk, W., Muller, F., Kellner, R., and Koch, K.-W. (1996) J. Biol. Chem. 271, 8022–8027). We demonstrate that the N terminus of GCAP-2, like those of other members of the recoverin family of Ca2+-binding proteins, is fatty acylated. However, unlike other proteins of this family, more GCAP-2 is present in the membrane fraction at low Ca2+ than at high Ca2+ concentrations. We investigated the role of the N-terminal fatty acyl residue in the ability of GCAP-2 to regulate RetGCs. Myristoylated or nonacylated GCAP-2 forms were expressed inEscherichia coli. Wild-type GCAP-2 and the Gly2→ Ala2 GCAP-2 mutant, which is unable to undergo N-terminal myristoylation, were also expressed in mammalian HEK293 cells. We found that compartmentalization of GCAP-2 in photoreceptor outer segment membranes is Ca2+- and ionic strength-sensitive, but it does not require the presence of the fatty acyl group and does not necessarily directly reflect GCAP-2 interaction with RetGC. The lack of myristoylation does not significantly affect the ability of GCAP-2 to stimulate RetGC. Nor does it affect the ability of the Ca2+-loaded form of GCAP-2 to compete with the GCAP-2 mutant that constitutively activates RetGC. We conclude that while Ca2+ binding plays a major regulatory role in GCAP-2 function, it does not operate through a calcium-myristoyl switch similar to the one found in recoverin.

THE MEMBRANE GUANYLYL CYCLASE, RETINAL GUANYLYL CYCLASE-1, IS ACTIVATED THROUGH ITS INTRACELLULAR DOMAIN

Retinal guanylyl cyclase-1 (RetGC-1) is a membrane guanylyl cyclase found in photoreceptor outer segments. It consists of an apparent extracellular domain (ECD) linked by a single transmembrane segment to an intracellular domain (ICD). Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca-binding protein that activates RetGC-1 in a Ca-sensitive manner. To establish whether GCAP-2 stimulates RetGC-1 through the ECD or ICD, we made deletion mutants lacking either the ECD or both the ECD and transmembrane domains (TMD) of RetGC-1. Recombinant wild type RetGC-1 and both deletion mutants were expressed in HEK 293 cells, and their sensitivities to GCAP-2, Ca, and ATP were compared. Our data demonstrate that both deletion mutants are regulated similarly to wild type RetGC-1 with indistinguishable EC values for Ca and similar K values for activation by GCAP-2. This shows that GCAP-2 functions through the ICD of RetGC-1 and that removal of the ECD and TMD do not significantly alter regulation by these factors. Our data also show that ATP potentiates stimulation of guanylyl cyclase activity by GCAP-2 and that neither the ECD nor the TMD of RetGC-1 participate in its regulation by ATP.

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

Guanylyl cyclase-activating proteins (GCAPs) are Ca2+-binding proteins that activate guanylyl cyclase when free Ca2+ concentrations in retinal rods and cones fall after illumination and inhibit the cyclase when free Ca2+ reaches its resting level in the dark. Several forms of retinal dystrophy are caused by mutations in GUCA1A, the gene coding for GCAP1. To investigate the cellular mechanisms affected by the diseased state, we created transgenic mice that express GCAP1 with a Tyr99Cys substitution (Y99C GCAP1) found in human patients with a late-onset retinal dystrophy (Payne et al., 1998). Y99C GCAP1 shifted the Ca2+ sensitivity of the guanylyl cyclase in photoreceptors, keeping it partially active at 250 nm free Ca2+, the normal resting Ca2+ concentration in darkness. The enhanced activity of the cyclase in the dark increased cyclic nucleotide-gated channel activity and elevated the rod outer segment Ca2+ concentration in darkness, measured by using fluo-5F and laser spot microscopy. In different lines of transgenic mice the magnitude of this effect rose with the Y99C GCAP1 expression. Surprisingly, there was little change in the rod photoresponse, indicating that dynamic Ca2+-dependent regulation of cGMP synthesis was preserved. However, the photoreceptors in these mice degenerated, and the rate of the cell loss increased with the level of the transgene expression, unlike in transgenic mice that overexpressed normal GCAP1. These results provide the first direct evidence that a mutation linked to congenital blindness increases Ca2+ in the outer segment, which may trigger the apoptotic process.

Inactivation of EF-hands Makes GCAP-2 (p24) a Constitutive Activator of Photoreceptor Guanylyl Cyclase by Preventing a Ca2+-induced “Activator-to-Inhibitor” Transition

Guanylyl cyclase activator proteins GCAP-1 and GCAP-2 (Dizhoor et al., 1995, Gorczyca et al., 1995) are members of a recently identified subclass of EF-hand type Ca2+-binding proteins that respond to Ca2+ differently than any other known members of the EF-hand superfamily. GCAPs acquire an activating conformation only in their Ca2+-free form. Free Ca2+ concentrations corresponding to levels in dark-adapted vertebrate photoreceptors inhibit the ability of GCAPs to activate photoreceptor guanylyl cyclases (RetGCs). We studied the effects of mutations that block binding of Ca2+ to the EF-hands of GCAP-2. Unlike other EF-hand proteins, which fail to activate their target when their EF-hands are inactivated by mutations, GCAP-2 with any single EF-hand inactivated remains active and is 3-6 times less sensitive to the inhibitory effect of Ca2+. Inactivation of any two or all three EF-hands produces active forms of GCAP-2 that are insensitive to inhibition by physiological intracellular concentrations of Ca2+. Unexpectedly we also found that activation of RetGCs by a Ca2+-insensitive mutant is inhibited by Ca2+-loaded wild type GCAP-2. We propose the following. 1) GCAP-2 can exist in two extreme functional forms: an apo form that activates RetGCs and a Ca2+-loaded form that blocks activation of RetGCs. 2) All three EF-hands of GCAP-2 contribute to the inhibitory effect of Ca2+. 3) Inactivation of two or three EF-hands is sufficient to shift the “activator-inhibitor” transition outside the physiological range of intracellular free Ca2+.