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The Function of Guanylate Cyclase 1 and Guanylate Cyclase 2 in Rod and Cone Photoreceptors

2007; Elsevier BV; Volume: 282; Issue: 12 Linguagem: Inglês

10.1074/jbc.m610369200

ISSN

1083-351X

Autores

Wolfgang Baehr, Sukanya Karan, Tadao Maeda, Dong-Gen Luo, Sha Li, J. Darin Bronson, Carl B. Watt, King-Wai Yau, Jeanne M. Frederick, Krzysztof Palczewski,

Tópico(s)

Photoreceptor and optogenetics research

Resumo

Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin α-subunit were mostly unaffected. Outer segment membranes of GC1–/– and GC double knock-out cones were destabilized and devoid of cone transducin (α- and γ-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the down-regulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins. Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin α-subunit were mostly unaffected. Outer segment membranes of GC1–/– and GC double knock-out cones were destabilized and devoid of cone transducin (α- and γ-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the down-regulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins. The second messenger for phototransduction in rods and cones of the vertebrate retina, cyclic GMP (cGMP), 2The abbreviations used are: cGMP, cyclic GMP; COS, cone outer segments; ERG, electroretinogram; GC, guanylate cyclase; GC1, guanylate cyclase 1 (synonymous with GC-E); GC2, guanylate cyclase 2 (synonymous with GC-F); GCAP, GC-activating protein; GCdko, GC double knock-out; GRK1, G protein-coupled receptor kinase 1; LCA, Leber congenital amaurosis; PDE6, photoreceptor cGMP phosphodiesterase 6; PDE6α′, cone PDE6 α subunit; ROS, rod outer segments; WT, wild type; pAb, polyclonal antibody; mAb, monoclonal antibody; cd, candela; CNG, cyclic nucleotide-gated; RT, reverse transcription.2The abbreviations used are: cGMP, cyclic GMP; COS, cone outer segments; ERG, electroretinogram; GC, guanylate cyclase; GC1, guanylate cyclase 1 (synonymous with GC-E); GC2, guanylate cyclase 2 (synonymous with GC-F); GCAP, GC-activating protein; GCdko, GC double knock-out; GRK1, G protein-coupled receptor kinase 1; LCA, Leber congenital amaurosis; PDE6, photoreceptor cGMP phosphodiesterase 6; PDE6α′, cone PDE6 α subunit; ROS, rod outer segments; WT, wild type; pAb, polyclonal antibody; mAb, monoclonal antibody; cd, candela; CNG, cyclic nucleotide-gated; RT, reverse transcription. is presumed to be synthesized by guanylate cyclases GC1 (1Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (237) Google Scholar) (GC-E) and GC2 (GC-F) and particulate enzymes and integral membrane proteins with a single transmembrane domain (1Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (237) Google Scholar, 2Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar, 3Yang R.B. Garbers D.L. J. Biol. Chem. 1997; 272: 13738-13742Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). GC1 is detected in the retina, pineal gland, and olfactory bulb (4Duda T. Koch K.W. Mol. Cell. Biochem. 2002; 230: 107-116Crossref PubMed Scopus (6) Google Scholar) as well as the cochlear nerve and the organ of Corti (5Seebacher T. Beitz E. Kumagami H. Wild K. Ruppersberg J.P. Schultz J.E. Hear. Res. 1999; 127: 95-102Crossref PubMed Scopus (27) Google Scholar), whereas GC2 is found only in the retina. The activities of these enzymes are Ca2+-sensitive, a sensitivity that is mediated by guanylate cyclase-activating proteins (GCAPs). GCAPs are Ca2+-binding proteins with three high affinity Ca2+-binding sites (EF hands) (6Palczewski K. Polans A.S. Baehr W. Ames J.B. Biol. Essays. 2000; 22: 337-350Crossref PubMed Scopus (135) Google Scholar, 7Dizhoor A.M. Hurley J.B. Methods. 1999; 19: 521-531Crossref PubMed Scopus (66) Google Scholar, 8Koch K.W. Adv. Exp. Med. Biol. 2002; 514: 349-360Crossref PubMed Scopus (12) Google Scholar). In light, the activation of cGMP phosphodiesterase (PDE6) in rods and cones leads to the hydrolysis of cGMP and the closure of cGMP-gated (CNG) cation channels (for a review, see Ref. 9Lamb T.D. Pugh Jr., E.N. Investig. Ophthalmol. Vis. Sci. 2006; 47: 5138-5152Crossref Scopus (227) Google Scholar). Channel closure causes the cytosolic free Ca2+ concentration to decline. The resulting dissociation of Ca2+ from the GCAPs increases the activity of the GCs and therefore the cGMP level, completing the negative feedback loop (10Burns M.E. Mendez A. Chen J. Baylor D.A. Neuron. 2002; 36: 81-91Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Thus, GCs are not only important for maintaining the basal cGMP level in darkness but are also critical for expediting the recovery of photoreceptors following light stimulation. Of the two GCAPs present in mouse retina, GCAP1 stimulates GC1 more efficiently than GC2 (11Haeseleer F. Sokal I. Li N. Pettenati M. Rao N. Bronson D. Wechter R. Baehr W. Palczewski K. J. Biol. Chem. 1999; 274: 6526-6535Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Deletion of both GCAP genes delays recovery of the dark current due to loss of Ca2+-dependent GC activation (12Mendez A. Burns M.E. Sokal I. Dizhoor A.M. Baehr W. Palczewski K. Baylor D.A. Chen J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9948-9953Crossref PubMed Scopus (212) Google Scholar). In addition to particulate GCs, a role in photoreceptor physiology was envisioned for soluble GCs, but no genetic or functional evidence is available for a role in modulation of cGMP in photoreceptors (13Noll G.N. Billek M. Pietruck C. Schmidt K.F. Neuropharmacology. 1994; 33: 1407-1412Crossref PubMed Scopus (35) Google Scholar, 14Cao L. Blute T.A. Eldred W.D. Vis. Neurosci. 2000; 17: 319-329Crossref PubMed Scopus (44) Google Scholar). In humans, null mutations in the GC1 gene (GUCY2D, on chromosome 17) are known to cause Leber congenital amaurosis type 1 (LCA1), an autosomal recessive, early onset rod/cone dystrophy (15Tucker C.L. Ramamurthy V. Pina A.L. Loyer M. Dharmaraj S. Li Y. Maumenee I.H. Hurley J.B. Koenekoop R.K. Mol. Vis. 2004; 10: 297-303PubMed Google Scholar, 16Hanein S. Perrault I. Gerber S. Tanguy G. Barbet F. Ducroq D. Calvas P. Dollfus H. Hamel C. Lopponen T. Munier F. Santos L. Shalev S. Zafeiriou D. Dufier J.L. Munnich A. Rozet J.M. Kaplan J. Hum. Mutat. 2004; 23: 306-317Crossref PubMed Scopus (284) Google Scholar). A naturally occurring retinal degeneration in chicken (rd or GUCY1*B chicken), caused also by a null mutation in the GC1 gene (17Semple-Rowland S.L. Lee N.R. Van Hooser J.P. Palczewski K. Baehr W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1271-1276Crossref PubMed Scopus (129) Google Scholar), mimics the human LCA phenotype. In mice, however, a null mutation in the GC1 gene produces a cone dystrophy; the GC1–/– rods remain viable and responsive to light (18Yang R.B. Robinson S.W. Xiong W.H. Yau K.W. Birch D.G. Garbers D.L. J. Neurosci. 1999; 19: 5889-5897Crossref PubMed Google Scholar, 19Coleman J.E. Zhang Y. Brown G.A. Semple-Rowland S.L. Investig. Ophthalmol. Vis. Sci. 2004; 45: 3397-3403Crossref PubMed Scopus (40) Google Scholar). No human retinal disease has yet been linked to a defect in the GC2 gene (GUCY2F, on the X chromosome). Here, the functional role of GC2 was examined by deleting the mouse GC2 gene. Our results show that electroretinographic (ERG) responses are largely normal in GC2–/– retina. In contrast, the ERG responses of GC1/GC2 double knock-out (GCdko) mice are absent, suggesting that no GC other than GC1 and GC2 is involved in rod or cone phototransduction. Histology of GCdko retinas revealed that outer segments form but degenerate in a manner similar to that observed in the rd (GUCY1*B) chicken (17Semple-Rowland S.L. Lee N.R. Van Hooser J.P. Palczewski K. Baehr W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1271-1276Crossref PubMed Scopus (129) Google Scholar) and in human LCA1 patients (20Rozet J.M. Perrault I. Gerber S. Hanein S. Barbet F. Ducroq D. Souied E. Munnich A. Kaplan J. Investig. Ophthalmol. Vis. Sci. 2001; 42: 1190-1192PubMed Google Scholar). Ultrastructural analyses detailed abnormal outer segment morphologies. Down-regulation of PDE6 and GCAPs in GCdko rods as well as down-regulation of cone phototransduction components in GC1–/– cones suggest that GCs serve enzymatic, stabilizing, and structural/transport roles in photoreceptors. Construction of Targeting Vector and Generation of GC2 Knock-out Mice—To delete the GC2 gene, a 16-kb mouse genomic DNA fragment containing exon 1 was cloned from a mouse 129Sv/Ev λ genomic library. An 8.5-kb XbaI fragment was subcloned to produce the targeting vector. One side of the neo gene cassette was inserted 5 bp upstream of the ATG start codon in exon 2. The replaced sequence starts with 5′-AGGCTATGTTCCTCGGACCCTGGCCTTTTTCTCGCC. The other side of the neo gene cassette was inserted 290 bp downstream of the ATG start codon inside exon 2, and that replaced sequence reads 5′-ATTCGAAGGAGTAACTCCTGTCAAATGTCTTGTCCCGG. In this strategy, the neo cassette replaced part of the coding region of exon 2. Ten micrograms of the targeting vector were linearized by NotI and then transfected by electroporation of iTL1 ES cells (129Sv/Ev) embryonic stem cells. After selection in G418, surviving colonies were expanded; PCR analysis was performed using primer pair Gctoda6 and PLA2 to identify recombinant clones. Primer Gctoda6 is located 165 bp downstream of the XbaI site, on the short arm side, with a sequence of 5′-GTTCTGAGCTACAGATCCTACAGTG. Primer PLA2 is located in the 5′-promoter region of the neo gene cassette and has a sequence of 5′-GTTCTTCGGACGCCTCGTCAACAC. The positive clones gave rise to a 1.5-kb PCR fragment. Correctly targeted ES cell lines were microinjected into C57BL/6J blastocysts. Chimeric mice were generated and gave germ line transmission of the GC2 knock-out to progeny. Primer pair Gctoda6 and Gctowt1 was used to identify the WT allele by its 1.3-kb amplified product. Primer Gctowt1 is located 280 bp downstream of the ATG start codon inside exon 1, inside the neo cassette replaced area, with a sequence of 5′-CAAGACATTTGACAGGAGTTACTC. Alternatively, primer Gctowt1 can be exchanged for primer Gctowt2, which is located 6 bp upstream of primer Gctowt1, with a sequence of 5′-CTGCTTTAGCAATTGAGCGAATCAG. Amplification yields a 1.3-kb fragment. PCR conditions were 94 °C for 20 s, 62 °C for 60 s, and 72 °C for 120 s (35 cycles). In difficult amplifications, 10% Me2SO or Q solution from Qiagen was used. GC1–/– and Double Knock-out Mice—Procedures for the animal experiments described here were IACUC-approved by the University of Utah and Case Western Reserve University and conformed to recommendations of the Association of Research for Vision and Ophthalmology. Animals were maintained in complete darkness, or cyclic light (12 h light/12 h dark or 14 h light/10 h dark) conditions, and physiological experiments were performed under dim red illumination using a Kodak number 1 Safelight filter (transmittance >560 nm). GC1 knock-out mice were rederived from GC1 knock-outs (19Coleman J.E. Zhang Y. Brown G.A. Semple-Rowland S.L. Investig. Ophthalmol. Vis. Sci. 2004; 45: 3397-3403Crossref PubMed Scopus (40) Google Scholar) using mice originally produced by Dr. David Garbers (University of Texas Southwestern, Dallas, TX) (18Yang R.B. Robinson S.W. Xiong W.H. Yau K.W. Birch D.G. Garbers D.L. J. Neurosci. 1999; 19: 5889-5897Crossref PubMed Google Scholar). GC single and double knock-outs were typed for the presence of GC1 and GC2 knock-out alleles and absence of WT alleles. Primers used for GC1 genotyping were as follows: Gc1F4 (forward primer in intron 4), 5′-TCCTATCCACGACAGGACCAAGACTGT; Gc1R4 (reverse primer in intron 5), 5′-GAGAGCAGAAGGGTAGCATTAGCTCAG; NeoF4 (forward primer in neo cassette), 5′-ACCGCTATCAGGACATAGCGTTGGCTA. Both pairs of primers were used in a one-tube PCR amplification to yield the fragments shown in Fig. 1D. GC2–/– mice were genotyped using forward primer Pla2 and reverse primer Gc2mt-R2 (5′-GTGCCTACAGGACTCTTGATGTCATTC). WT mice were genotyped with primers Gctowt1 and Gctoda6. Antibodies—The following laboratories have generously provided antibodies: David Garbers, University of Texas South-western (pAb L670, anti-GC2), Tiansen Li, Harvard University (anti-PDE6α′), Cheryl Craft, University of Southern California (anti-cone arrestin, mCAR or LUMIJ), Ching-Kang Chen, Virginia Commonwealth University (UUTA, anti-rod transducin α subunit), Robert Molday, University of British Columbia (1D4, anti-rhodopsin, and anti-CNGA1 subunit antibody), and Rick Cote, University of New Hampshire (anti-PrBP/δ (PDEδ-FL). The following antibodies were commercially available: anti-cone Tγ and anti-PDE6 (MOE, Cytosignal Research Products), anti-cone Tα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-S opsin and anti-M/L opsin (Chemicon/Millipore, Temecula, CA). Real Time RT-PCR—Semiquantitative real time RT-PCR was performed using total RNA of WT and GCdko retinas. The following primers were used: rod PDE6α subunit forward, 5′-TGATGAGTACGAAGCCAAGATGAAGGC; rod PDE6α subunit reverse, 5′-TCAGCTACTGGATGCAACAGGACTTAG; rod Tα subunit forward, 5′-ACGCTGTCACCGACATTATCATCAAGG; rod Tα subunit reverse, 5′-AGCAGCTTGTGGAAAGGACGGTATTTG; cone Tα subunit forward, 5′-ATGACCTGTGCTACAGACACACAGAAC; cone Tα subunit reverse, 5′-GCATGAAGCCTCAGATTCTAAGCTTGC; GCAP1 forward, 5′-AGTTGCGCTGGTATTTCAAGCTCTACG; GCAP1 reverse, 5′-AAACACGGTATCTGTGAATTCCTCGGC; GCAP2 forward, 5′-TATGTAGAGAGCATGTTCCGAGCCTTC; GCAP2 reverse, 5′-ATGCAGCCATTTCGGTCCTTGTCATAG; Gapdh forward, 5′-accccttcattgacctcaactacatgg; Gapdh reverse, 5′-atttgatgttagtggggtctcgctccT. All primer pairs were designed to give an amplicon size of ∼150 bp. Total RNAs were extracted from 1-month-old mouse retina by using Trizol reagent (Invitrogen) followed by DNase digestion. Real-time RT-PCR was performed with both WT and GCdko RNA samples (25 ng) using the QuantiTect SYBR green RT-PCR kit (Qiagen Inc., Valencia, CA) and a DNA Engine Opticon 2 system (MJ Research, Waltham, MA). Standard curves for each primer set were obtained with 0, 10, 20, 40, 80, and 160 ng of the WT RNA sample (Fig. S3). Results were normalized to Gapdh signals for each sample. Standards for Gapdh and all other primer sets showed similar slopes, indicating equivalent amplification kinetics (Fig. S3). Confocal Immunolocalization—Eyes were harvested at mid-morning under ambient illumination (200–800 lux) without dark or light adaptation. Left eyes of age-matched mice were immersion-fixed for 2 h using freshly prepared 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4) and cryoprotected. Immunocytochemistry was performed as described (21Bhosale P. Larson A.J. Frederick J.M. Southwick K. Thulin C.D. Bernstein P.S. J. Biol. Chem. 2004; 279: 49447-49454Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Affinity-purified primary antibodies (15–25 μl) were applied to each group of four sections in a humidified, rotating chamber overnight at 4 °C. Propidium iodide (Invitrogen Molecular Probes™; 1:3,000 dilution) was added to the solution containing fluorescein isothiocyanate-conjugated secondary antibody. The sections were viewed using a Zeiss LSM 510 inverted Laser Scan confocal microscope with a ×40, 1.3 numerical aperture oil objective lens and optical slit setting of <0.9 μm. The following antibodies and dilutions were used: anti-GC1 (mAb 1S4, 1:1,000) (22Haire S.E. Pang J. Boye S.L. Sokal I. Craft C.M. Palczewski K. Hauswirth W.W. Semple-Rowland S.L. Investig. Ophthalmol. Vis. Sci. 2006; 47: 3745-3753Crossref PubMed Scopus (64) Google Scholar); anti-GC2 (L-670, 1:2000); anti-GCAP1 (pAb UW14, 1:1,000) (23Pennesi M.E. Howes K.A. Baehr W. Wu S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6783-6788Crossref PubMed Scopus (61) Google Scholar); anti-GCAP2 (pAb UW50, 1:4,000) (24Otto-Bruc A. Fariss R.N. Haeseleer F. Huang J. Buczylko J. Surgucheva I. Baehr W. Milam A.H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4727-4732Crossref PubMed Scopus (82) Google Scholar); anti-rhodopsin (mAb 1D4, 1:1,000); anti-rod Tα (UUTA, 1:1,000); anti-rod PDE6 (MOE, 1:1,000); anti-S opsin (1:500); anti-M/L opsin (1:500); anti-cone arrestin (mCAR, 1:1,000); anti-cone PDE6α′ (1:4,000); anti-cone Tα (1:500); anti-cone Tγ (1:500); anti-GRK1 (G8, 1:800) (25Zhao X. Huang J. Khani S.C. Palczewski K. J. Biol. Chem. 1998; 273: 5124-5131Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Immunoblotting—Retinas from both eyes of the mouse were sonicated in 100 μl of lysis buffer (26Howes K.A. Bronson J.D. Dang Y.L. Li N. Zhang K. Ruiz C.C. Helekar B.S. Lee M. Subbaraya I. Kolb H. Chen J. Baehr W. Investig. Ophthalmol. Vis. Sci. 1998; 39: 867-875PubMed Google Scholar). Each sample (15 μg of protein) was separated on a 10–12% SDS-PAGE, transferred onto a polyvinylidene difluoride filter, and probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody. The signal was visualized using chemiluminescence (ECL Plus system (Amersham Biosciences). ERGs—The recording procedure was performed as described (27Haeseleer F. Imanishi Y. Maeda T. Possin D.E. Maeda A. Lee A. Rieke F. Palczewski K. Nat. Neurosci. 2004; 7: 1079-1087Crossref PubMed Scopus (227) Google Scholar, 28Maeda A. Maeda T. Imanishi Y. Kuksa V. Alekseev A. Bronson J.D. Zhang H. Zhu L. Sun W. Saperstein D.A. Rieke F. Baehr W. Palczewski K. J. Biol. Chem. 2005; 280: 18822-18832Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). ERGs were recorded with the universal testing and electrophysiologic system UTAS E-3000 (LKC Technologies, Inc.). The light intensity was calibrated by the manufacturer and computer-controlled. Flash stimuli had a range of intensities (–3.7-2.8 log cd·s·m–2), and white light flash duration was adjusted according to intensity (from 20 μs to 1 ms). Leading edges of the ERG responses were fitted with a model of rod photoreceptor activation (29Van Hooser J.P. Aleman T.S. He Y.G. Cideciyan A.V. Kuksa V. Pittler S.J. Stone E.M. Jacobson S.G. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8623-8628Crossref PubMed Scopus (226) Google Scholar). The double-flash recording followed a previously published protocol (30Liang Y. Fotiadis D. Maeda T. Maeda A. Modzelewska A. Filipek S. Saperstein D.A. Engel A. Palczewski K. J. Biol. Chem. 2004; 279: 48189-48196Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The normalized amplitude of the probe flash a-wave versus the time between two flashes was plotted and fit by the linear regression algorithm in the SigmaPlot program (version 9.0). Dark-adapted ERG recordings after intense constant illumination were performed and evaluated as described (28Maeda A. Maeda T. Imanishi Y. Kuksa V. Alekseev A. Bronson J.D. Zhang H. Zhu L. Sun W. Saperstein D.A. Rieke F. Baehr W. Palczewski K. J. Biol. Chem. 2005; 280: 18822-18832Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 31Kim T.S. Maeda A. Maeda T. Heinlein C. Kedishvili N. Palczewski K. Nelson P.S. J. Biol. Chem. 2005; 280: 8694-8704Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Single Cell Recordings—Single cell recordings were performed as described previously (32Luo D.G. Yau K.W. J. Gen. Physiol. 2005; 126: 263-269Crossref PubMed Scopus (24) Google Scholar). An individual rod outer segment was drawn into a snug-fitting glass suction electrode containing 140 mm NaCl, 3.6 mm KCl, 2.4 mm MgCl2, 1.2 mm CaCl2, 3 mm HEPES, pH 7.4, 0.02 mm EDTA, and 10 mm glucose. Membrane current was monitored with a current-to-voltage amplifier (Axopatch 200B, Axon Instruments, CA). All signals were low pass-filtered at 20 Hz (8-pole Bessel) and sampled at 500 Hz. The light stimulus consisted of brief flashes (10 ms) of unpolarized 500-nm (10-nm bandwidth) light with intervals of 8 s. Histology—Right eyes of 1–2-month-old WT, GC1–/–, GC2–/–, and GCdko mice were prepared for light microscope plastic sections and electron microscope thin sections as described (33Frederick J.M. Krasnoperova N.V. Hoffmann K. Church-Kopish J. Ruether K. Howes K.A. Lem J. Baehr W. Investig. Ophthalmol. Vis. Sci. 2001; 42: 826-833PubMed Google Scholar). For electron microscopy, the eyecups were postfixed in 1% osmium tetroxide in phosphate buffer for 1 h, dehydrated through an ascending series of graded ethanols, and embedded in Spurr's resin. Sections 0.5–1 μm thick and passing through the optic nerve were imaged using a Leica DM-R microscope with Prior stage, using Syncroscan RT software from Syncroscopy. Scaling for measurement was 182 nm/pixel and 5.5 pixels/micrometer at ×40 magnification. Generation of GC2–/– and GC1/GC2 Double Knock-out (GCdko) Mice—The GC1 (Gucy2e, on mouse chromosome 11) and the GC2 (Gucy2f, on the X chromosome) genes are closely related in structure (Fig. 1, A and B) (34Yang R.B. Fulle H.J. Garbers D.L. Genomics. 1996; 31: 367-372Crossref PubMed Scopus (45) Google Scholar). The GC1 knock-out construct (18Yang R.B. Robinson S.W. Xiong W.H. Yau K.W. Birch D.G. Garbers D.L. J. Neurosci. 1999; 19: 5889-5897Crossref PubMed Google Scholar) replaced a portion of exon 5 encoding the transmembrane domain by a neo cassette, thereby deleting the entire intracellular region of GC1. We prepared a GC2 knock-out mouse by replacing a portion of exon 2 containing the translation start codon and the peptide leader sequence with a neo cassette (Fig. 1, B and C). Several generations of these mice were established on the C57BL/6J background and were cross-bred with GC1–/– mice to generate GCdko mice. The GCdko mice were fertile and developed normally, thereby excluding a vital role for GC1 or GC2 during embryogenesis. The genotypes of single and double knock-out mice were confirmed by PCR amplification using GC1- and GC2-specific oligonucleotides (Fig. 1D). Immunoblots of GC1–/– and GC2–/– retinal lysates probed with monoclonal anti-GC1 and polyclonal anti-GC2 antibodies confirmed that GC1 and GC2 were not expressed (Fig. 1E). Further, immunoblots with anti-GC1 and anti-GC2 antibodies showed that the GC1 expression level is maintained in GC2–/– retina and that the GC2 expression level is also maintained in GC1–/– retina (Fig. 1F). GCdko Rod and Cone Photoreceptors Are Physiologically "Silent"—To test for photoreceptor function in WT and single and double knock-out retinas, we performed full-field ERGs (Table 1 and Fig. 2). Consistent with a previous report (18Yang R.B. Robinson S.W. Xiong W.H. Yau K.W. Birch D.G. Garbers D.L. J. Neurosci. 1999; 19: 5889-5897Crossref PubMed Google Scholar), scotopic a- and b-wave amplitudes recorded from 4–6-week-old GC1–/– mice were significantly reduced compared with WT responses (Fig. 2, A and B). However, scotopic ERG responses were absent in GCdko mice at all light intensities tested. These results suggest that the rod responses recorded from GC1–/– retinas reflect GC2 activity. Scotopic ERG responses recorded from GC2–/– mice closely resembled those of WT mice, implying that GC1 alone is able to support rod function under these conditions. The a-wave amplitude and sensitivity of the scotopic GC2–/– mouse photoresponse were similar to WT at low intensities (Fig. 2B; Table 1); however, at high intensities, GC2–/– a-wave amplitudes were slightly reduced compared with WT (Fig. 2B). Photopic ERG responses recorded in the presence of background light were absent in GC1–/– and GCdko mice, whereas GC2–/– a- and b-waves were nearly identical to WT (Fig. 2, C and D). These results are consistent with GC1 regulating cGMP synthesis in cones and with both GC1 and GC2 regulating cGMP synthesis in rods. Given these observations, the possibility that other cyclases participate in rod or cone phototransduction can be excluded.TABLE 1ERG data (a-wave amplitudes, sensitivity, and recovery) for WT, GC1-/-, and GC2-/- miceWTGC1-/-GC2-/-Maximum a-wave amplitude (μV)964.6 ± 48.4239.3 ± 50.5ap < 0.0001.874.8 ± 23.9Sensitivity (log scotopic-cd-1 m2 s-3)6.2 ± 0.36.4 ± 1.24.4 ± 0.4bp < 0.05.Time between flashes for 50% a-wave amplitude recovery (ms)893.3 ± 47.8895.4 ± 112.21,018.1 ± 63.1a p < 0.0001.b p < 0.05. Open table in a new tab Recovery from Test Flashes as Determined by Paired Flash Analysis—A paired flash protocol was used to evaluate the ability of GC deletion mutant retinas to recover responsiveness after light stimulation (23Pennesi M.E. Howes K.A. Baehr W. Wu S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6783-6788Crossref PubMed Scopus (61) Google Scholar, 30Liang Y. Fotiadis D. Maeda T. Maeda A. Modzelewska A. Filipek S. Saperstein D.A. Engel A. Palczewski K. J. Biol. Chem. 2004; 279: 48189-48196Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 35Howes K.A. Pennesi M.E. Sokal I. Church-Kopish J. Schmidt B. Margolis P. Frederick J.M. Rieke F. Palczewski K. Wu S.M. Detwiler P.B. Baehr W. EMBO J. 2002; 21: 1545-1554Crossref PubMed Scopus (92) Google Scholar). In this method, short test flashes followed by varying interstimulus intervals, and a probe flash were used to test for a-wave recovery (36Pepperberg D.R. Birch D.G. Hood D.C. Vis. Neurosci. 1997; 14: 73-82Crossref PubMed Scopus (80) Google Scholar, 37Lyubarsky A.L. Pugh Jr., E.N. J. Neurosci. 1996; 16: 563-571Crossref PubMed Google Scholar). As expected, GCdko photoreceptors were unresponsive (Fig. 3A). Recoveries of a-waves measured from GC1–/– mice and GC2–/– mice were normalized to the amplitudes of corresponding WT a-wave responses. Rates of dark current recovery in GC1–/– and GC2–/– mice after the test flash were not significantly different from WT mice (GC1–/–, 895.4 ± 112.2 ms; GC2–/–, 1018.1 ± 63.1 ms; WT, 893.3 ± 47.8 ms) (Table 1). Intense Bleach Delays Recovery of GC1–/– and GC2–/– a-wave Amplitudes—In a second approach, dark adaptation rates were determined by exposing animals to a high intensity bleaching light (500 cd·s·m–2) for 3 min and then recording a-wave responses elicited by probe flashes delivered intermittently over the course of a 60-min dark adaptation period (Fig. 3B). Under these conditions, 70% of rhodopsin was bleached (28Maeda A. Maeda T. Imanishi Y. Kuksa V. Alekseev A. Bronson J.D. Zhang H. Zhu L. Sun W. Saperstein D.A. Rieke F. Baehr W. Palczewski K. J. Biol. Chem. 2005; 280: 18822-18832Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The ratios of the a-wave responses obtained during the recovery phase following the bleach for WT, GC1–/–, GC2–/–, and GCdko mice were plotted as a function of time in darkness. In WT mice, a-wave amplitudes recovered about 70% after 1 h of dark adaptation. The recovery of a-wave amplitudes was significantly delayed in GC1–/– and GC2–/– mice (p < 0.001) compared with WT. Following 1 h of dark adaptation, the amplitudes of the a-waves recorded from GC2–/– and GC1–/– mice had recovered to about 30 and 50%, respectively, of their dark-adapted values. These results suggest that GC1 and GC2 contribute distinctly to the recovery of photoreceptors from exposure to intense bleaching light. Rod Photoreceptor Single Cell Recordings—The mean dark currents of GC1–/– and GC2–/– rod photoreceptors were not significantly different from the WT currents (12 ± 1.2 pA for WT, 12.7 ± 1.3 pA for GC1–/–, and 13.8 ± 1.3 pA for GC2–/–) (Fig. 4, A–C). In contrast, GCdko rods showed no detectable light-sensitive current (Fig. 4D). That cyclic nucleotidegated (CNG) channels are present at similar densities in the rod outer segments (ROS) of WT and GC single knock-out rods argues that [cGMP]i levels in darkness are similar for WT, GC1–/–, and GC2–/– rods, whereas effectively no cGMP is present in GCdko rods. It therefore follows that the basal activity of either GC1 or GC2 is sufficient to maintain dark current in rods. Compensatory up-regulation of the other GC when one is missing may account for this observation; however, immunoblot results suggest normal GC2 levels in GC1–/– mice (Fig. 1F) (18Yang R.B. Robinson S.W. Xiong W.H. Yau K.W. Birch D.G. Garbers D.L. J. Neurosci. 1999; 19: 5889-5897Crossref PubMed Google Scholar). Regarding the light response, the kinetics was broadly similar for all three genotypes (Fig. 4, A–C). Sensitivity of GC2–/– rods was quite similar to that of WT rods (half-saturating flash intensity of 52.8 ± 4.9 versus 48.6 ± 6.3 photons μm–2), but the sensitivity of GC1–/– rods (half-saturating flash intensity of 13.6 ± 2.5 photons μm–2) was about 3.5-fold higher than WT (Fig. 4E) (see also Ref. 18Yang R.B. Robinson S.W. Xiong W.H. Yau K.W. Birch D.G. Garbers D.L. J. Neurosci.

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