GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice
2002; Springer Nature; Volume: 21; Issue: 7 Linguagem: Inglês
10.1093/emboj/21.7.1545
ISSN1460-2075
Autores Tópico(s)Receptor Mechanisms and Signaling
ResumoArticle1 April 2002free access GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice Kim A. Howes Kim A. Howes Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Search for more papers by this author Mark E. Pennesi Mark E. Pennesi Department of Ophthalmology and Division of Neuroscience, Houston, TX, 77030 USA Search for more papers by this author Izabela Sokal Izabela Sokal Departments of Ophthalmology, Seattle, WA, 98195 USA Search for more papers by this author Jill Church-Kopish Jill Church-Kopish Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Search for more papers by this author Ben Schmidt Ben Schmidt Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Search for more papers by this author David Margolis David Margolis Departments of Physiology and Biophysics, Seattle, WA, 98195 USA Search for more papers by this author Jeanne M. Frederick Jeanne M. Frederick Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Search for more papers by this author Fred Rieke Fred Rieke Departments of Physiology and Biophysics, Seattle, WA, 98195 USA Search for more papers by this author Krzysztof Palczewski Krzysztof Palczewski Departments of Ophthalmology, Seattle, WA, 98195 USA Departments of Pharmacology and Chemistry, University of Washington, Seattle, WA, 98195 USA Search for more papers by this author Samuel M. Wu Samuel M. Wu Department of Ophthalmology and Division of Neuroscience, Houston, TX, 77030 USA Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, 77030 USA Search for more papers by this author Peter B. Detwiler Peter B. Detwiler Departments of Physiology and Biophysics, Seattle, WA, 98195 USA Search for more papers by this author Wolfgang Baehr Corresponding Author Wolfgang Baehr Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Departments of Biology, and Neurobiology and Anatomy, University of Utah, Salt Lake City, UT, 84112 USA Search for more papers by this author Kim A. Howes Kim A. Howes Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Search for more papers by this author Mark E. Pennesi Mark E. Pennesi Department of Ophthalmology and Division of Neuroscience, Houston, TX, 77030 USA Search for more papers by this author Izabela Sokal Izabela Sokal Departments of Ophthalmology, Seattle, WA, 98195 USA Search for more papers by this author Jill Church-Kopish Jill Church-Kopish Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Search for more papers by this author Ben Schmidt Ben Schmidt Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Search for more papers by this author David Margolis David Margolis Departments of Physiology and Biophysics, Seattle, WA, 98195 USA Search for more papers by this author Jeanne M. Frederick Jeanne M. Frederick Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Search for more papers by this author Fred Rieke Fred Rieke Departments of Physiology and Biophysics, Seattle, WA, 98195 USA Search for more papers by this author Krzysztof Palczewski Krzysztof Palczewski Departments of Ophthalmology, Seattle, WA, 98195 USA Departments of Pharmacology and Chemistry, University of Washington, Seattle, WA, 98195 USA Search for more papers by this author Samuel M. Wu Samuel M. Wu Department of Ophthalmology and Division of Neuroscience, Houston, TX, 77030 USA Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, 77030 USA Search for more papers by this author Peter B. Detwiler Peter B. Detwiler Departments of Physiology and Biophysics, Seattle, WA, 98195 USA Search for more papers by this author Wolfgang Baehr Corresponding Author Wolfgang Baehr Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA Departments of Biology, and Neurobiology and Anatomy, University of Utah, Salt Lake City, UT, 84112 USA Search for more papers by this author Author Information Kim A. Howes1, Mark E. Pennesi2, Izabela Sokal3, Jill Church-Kopish1, Ben Schmidt1, David Margolis4, Jeanne M. Frederick1, Fred Rieke4, Krzysztof Palczewski3,5, Samuel M. Wu2,6, Peter B. Detwiler4 and Wolfgang Baehr 1,7 1Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330 USA 2Department of Ophthalmology and Division of Neuroscience, Houston, TX, 77030 USA 3Departments of Ophthalmology, Seattle, WA, 98195 USA 4Departments of Physiology and Biophysics, Seattle, WA, 98195 USA 5Departments of Pharmacology and Chemistry, University of Washington, Seattle, WA, 98195 USA 6Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, 77030 USA 7Departments of Biology, and Neurobiology and Anatomy, University of Utah, Salt Lake City, UT, 84112 USA ‡K.A.Howes and M.E.Pennesi contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1545-1554https://doi.org/10.1093/emboj/21.7.1545 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca2+ and cGMP. Ca2+ regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP by acting on a GC-activating protein (GCAP). While this action is critical for rapid termination of the light response, the GCAP responsible has not been identified. To test if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses, transgenic mice were generated that express only GCAP1 under the control of the endogenous promoter. Paired flash responses revealed a correlation between the degree of recovery of the rod a-wave and expression levels of GCAP1. In single cell recordings, the majority of the rods generated flash responses that were indistinguishable from wild type. These results demonstrate that GCAP1 at near normal levels supports the generation of wild-type flash responses in the absence of GCAP2. Introduction Vision begins with the conversion of light into an electrical response by the retina's photoreceptors (for a recent review see McBee et al., 2001). The receptor light response involves changes in two intracellular second messengers, cGMP and Ca2+. In darkness, high intracellular [cGMP] keeps cyclic nucleotide gated ion channels (CNG channels) open and a steady current (carried by Na+ and Ca2+) flows into the outer segment. Light activates a G-protein coupled enzyme cascade that stimulates cGMP-specific phosphodiesterase (PDE). The resulting fall in [cGMP] causes the CNG channels to close and decreases Ca2+ influx. Efflux of Ca2+ by Na+/Ca2+,K+ exchange persists, causing a fall in intracellular Ca2+. The fall in Ca2+ stimulates synthesis of cGMP by guanylate cyclase (GC) and leads to timely recovery of the light response. Here we examine the molecular mechanism responsible for the Ca2+-regulation of photoreceptor GC. The mammalian retina contains two particulate GCs (GC1 and GC2), and two different GC-activating proteins (GCAP1 and GCAP2). A third GCAP has been identified only in humans (Haeseleer et al., 1999) and zebrafish (Imanishi et al., 2002). In vitro, at low Ca2+, GCAP1 stimulates GC1 while GCAP2 stimulates both GC1 and GC2; both GCAPs are inactive when free Ca2+ is high (Haeseleer et al., 1999). The distribution of GC1 and GC2 in rods and cones appears to be species dependent (Yang et al., 1999; Imanishi et al., 2002). Immunocytochemistry in bovine and mouse retina has shown that GCAP1 is present in rods and cones, while GCAP2 is seen nearly exclusively in rods (Cuenca et al., 1998; Kachi et al., 1999). The genes encoding GCAP1 and GCAP2 are arranged in an autosomal tail-to-tail array (Howes et al., 1998). This arrangement allows for generation of a double knockout mouse (GCAP−/−) in which expression of both GCAPs is disabled. Rod outer segment (ROS) membranes from GCAP−/− mice show no Ca2+ regulation of GC activity. The Ca2+ insensitivity of cGMP synthesis causes the flash response in these rods to be larger and slower than in wild type (Mendez et al., 2001). The Ca2+ dependence of GC activity is rescued by overexpression of bovine GCAP2 in GCAP−/− rods and the time for rod recovery from saturating flashes is returned to normal (Mendez et al., 2001). Overexpression of GCAP2 does not, however, restore the normal kinetics of responses evoked by subsaturating flashes. This suggests that GCAP1, either by itself or in conjunction with GCAP2, participates in the recovery of the rod flash response. To investigate the role of GCAP1 in rod phototransduction, transgenic mice were generated and bred with GCAP−/− mice to produce a mouse that expressed GCAP1 under the control of its endogenous promoter on a GCAP-null background. We present evidence that GCAP1 expression in GCAP−/− rods can restore the wild-type properties of the rod light response in the absence of GCAP2. Results Generation of transgenic mice expressing GCAP1 To evaluate the role of GCAP1 in phototransduction, we generated mice expressing GCAP1 under the control of the endogenous promoter. A transgene containing the entire mouse GCAP1 gene, including the polyadenylation site and a 5.4 kb upstream regulatory region (Figure 1A), was microinjected into single-cell embryos. Four GCAP1 transgenic (G1T) founder mice were generated, two of which (G1T3+ and G1T4+) passed the transgene to F1 offspring with 50% penetrance, as expected from genetically non-mosaic mice. Lines could not be established from the other two G1T founders. Southern blots showed that the G1T3+ founder contained approximately two to three copies, and the G1T4+ founder three to five copies of the transgene (data not shown). Figure 1.Gene constructs and promoter analysis of the G1T+ transgenic lines. (A) Wild-type GCAP gene array, disrupted gene array and GCAP1 transgene construct. The arrows above the GCAP array indicate the direction of transcription (the mGCAP1 and mGCAP2 genes are located on opposite strands). The specific fragments used for genotyping (wild-type fragment 1, neo fragment 3 and transgene fragment 2, see also Figure 2A) are shown as bars below the constructs (for details of sense and antisense primer pairs, see Materials and methods). Black boxes depict GCAP1 exons, gray boxes GCAP2 exons. Relevant restriction sites used for the constructs are: H, HindIII; X, XhoI; E, EcoRI. Hatched boxes indicate multiple cloning site sequences derived from the original cloning vector. (B) Amplified fragments of the GCAP1 promoter region indicating the presence and absence of a 1.8 kb deletion. Primers corresponding to sequences 0.5–3 kb upstream of ATG (in 500 bp increments) together with an anchoring antisense primer located at −170 were used to amplify upstream regulatory fragments. GCAP+/+ and the G1T4+GCAP+/+ transgenic line show the expected 3.0 kb product when amplified with the anchoring primer and primer −3379. The G1T3+GCAP+/+ transgenic line exhibits an additional band of 1.4 kb. This band was derived from the G1T3 promoter region from which 1.8 kb were deleted. (C) Schematic depiction of the G1T promoter region and identification of the deletion in the upstream region of the G1T3+ line. The wild-type promoter region contains two repeats (a and b), which are developmentally regulated by retinoic acid, a retinoic acid enhancer element (c) on the antisense strand, and an overlapping AP1 enhancer element (d) on the sense strand in a region between −1337 and −3201 (−1 is the first nucleotide upstream of the translation initiator ATG). This region, identified by a gray bar and marked as Δ, is deleted in the G1T3+ line. Download figure Download PowerPoint Irregularities in expression patterns (see below) prompted us to examine more closely both transgenes in offspring of the two founders. Analysis of the G1T3 transgene by primers generated in 500 bp increments revealed a deletion in the upstream region (Figure 1B and C). The deletion removes an ∼1.8 kb fragment 1337 bp upstream of the translation start codon, such that the sequence at position −1337 bp was contiguous with the sequence at −3201 bp. The deletion was seen consistently in PCRs of tail DNA from all G1T3+ mice assayed. No alterations were seen in the transgene present in G1T4+ mice, suggesting that the G1T3 upstream deletion occurred during integration of the transgene into the mouse genome by an unknown mechanism. The transcription start point of the mouse GCAP1 gene is predicted to be 294 bp upstream of ATG [by eukaryotic neural network promoter prediction (Ohler et al., 1999) and 5′ RACE; results not shown], and a putative TATA box is present 28 bp upstream of the transcription start point. Proximal regulatory elements, located within a 121 bp region, are highly conserved between the human and mouse GCAP1 genes. Within this region, retinoic acid and/or thyroid hormone, AP1, possibly RET1 and CRX responsive consensus elements are found, consistent with other photoreceptor-expressed genes. More distally, the mouse promoter region contains a large unsequenceable (CA)n repeat (position >–3.7 kb), which spans >500 bp. A similar but much smaller microsatellite sequence consisting of a (GA)n repeat is present in the first intron. The two transgenic lines, one with an intact promoter (G1T4+) and one with a partial deletion (G1T3+), provide the desired tools to determine whether GCAP1 can restore normal rod function and to analyze the consequences of the deletion for effects on GCAP1 expression levels and cellular distribution. Generation of transgenic mice expressing GCAP1 on a GCAP−/−background GCAP−/− mice were generated recently by replacing C-terminal exons of the GCAP1/2 gene array with a neo cassette (Figure 1A) (Mendez et al., 2001). When raised under normal light conditions, the null mice were shown to have normal retinal morphology and reduced a-wave electroretinogram (ERG) responses under dark-adapted conditions (Hurley and Chen, 2001). However, light responses of single GCAP−/− rods showed a prolonged rising phase of the light response and a pronounced slowing of the recovery phase (Mendez et al., 2001), consistent with an absence of the Ca2+-dependent stimulation of cGMP synthesis that normally accelerates the recovery process. Crossbreeding of G1T4+GCAP+/− mice with GCAP+/− mice generates offspring expressing GCAP1 on a null background (G1T4+GCAP−/−), as well as five other genotypes (Figure 2A). A corresponding set of six genotypes was also generated using the G1T3+ transgenic line. Genotyping of all three alleles (wild-type GCAP array, disrupted gene array and GCAP1 transgene) was performed simultaneously by PCR amplification, yielding diagnostic fragments of approximately 800, 200 and 500 bp, respectively (Figure 2A). Expression levels for the GCAP1 transgenes were determined with several western blots using G1T3+ and G1T4+ on the null background and comparison with GCAP1 signals in GCAP+/+ mice. G1T3+GCAP−/− transgenic retinas always showed ∼10–30% of wild-type GCAP1 expression, and GCAP1 expression in G1T4+GCAP−/− lines was ∼30–50% that of wild-type levels (Figure 2B). Figure 2.Genotyping of transgenic lines and analysis of GCAP1 expression. (A) Mobilities of diagnostic amplicons 1–3 for the various genotypes as outlined schematically in Figure 1A. The fragments were generated in single PCRs containing all primers using genomic DNA isolated from tail biopsies. The amplicons were separated on 1% agarose gels. The control lane contains the three primer pairs, but no genomic template. Fragment 1 is diagnostic of the wild-type GCAP array, fragment 2 of the G1T transgene (both G1T3+ and G1T4+ lines), and fragment 3 for the knockout construct. Genomic DNA amplifying only fragment 1 is GCAP+/+, genomic DNA amplifying fragment 3 is GCAP−/−, while DNA amplifying both fragments 1 and 3 is GCAP+/−. Correspondingly, all lines amplifying fragment 2 carry the G1T transgene. (B) Western blot of retinal homogenates. Polyclonal antibody UW14 (Gorczyca et al., 1995) was used to identify GCAP1 transgene expression in G1T3+/GCAP−/− and G1T4+/GCAP−/− retinas relative to wild-type levels. Retinal protein (10 μg) was run for each sample. The slightly faster mobility of GCAP1 in the G1T3+GCAP−/− retina sample was seen consistently in many western blots. Faster mobilities may be caused by the different amounts of calcium bound (Li et al., 2001) or by differential acylation of the GCAP1 protein (Sokal et al., 2001) in cones, since the G1T3 line shows predominantly cone expression. WT, wild type. Download figure Download PowerPoint GC stimulation levels at low Ca2+ were found consistently to be in between those for knockout and wild-type retinas (data not shown). Expression levels of 30–50% are consistent with expression of a heterozygous transgene (homozygous transgenics were not generated) and represent an average expression level in all photoreceptors. However, as shown below, expression in individual rods varies significantly. Cellular distribution of GCAP1 in GCAP1 transgenic lines GCAP1 transgene expression in photoreceptors of G1T3+ and G1T4+ lines on the wild-type background were determined by immunocytochemistry (Figure 3) and by functional analyses (Figures 4, 5, 6). To identify cellular and subcellular locations of transgenic GCAP1, we performed immunocytochemistry with a GCAP1-specific polyclonal antibodies (UW101 and UW14) on retinas collected from the nine genetic variations produced in this study (Figure 3A–I). As expected, GCAP−/− retinas were immunonegative for GCAP1 (Figure 3G). G1T lines on a GCAP−/− background were immunopositive for GCAP1 and immunonegative with the anti-GCAP2 antibody (data not shown) as opposed to wild-type GCAP2 staining. GCAP1 transgene expression was specifically targeted to photoreceptor cells in both G1T3+ and G1T4+ lines. The results are consistent with the ability of the intact endogenous GCAP1 promoter (5.4 kb) to direct expression of GCAP1 to both rods and cones. The G1T3+ GCAP+/+ and G1T4+GCAP+/+ transgenic mice (Figure 3B and C) showed additional GCAP1 expression, seen as intense immunoreactive packets in the outer segments, particularly in cones, relative to wild-type levels. Interestingly, GCAP1 immunolocalization was quite variable across photoreceptor outer segments in all lines except those expressing near normal GCAP1 levels, including the wild-type and G1T4+GCAP+/− mice. Figure 3.Immunocytochemical localization of GCAPs in retinas from all genotypes generated. GCAP1 was identified with polyclonal antibody UW101 by indirect immunofluorescence (FITC), and mouse cones were labeled with rhodamine-conjugated peanut agglutinin (PNA). The first row (A, D, G) contains GCAP+/+, GCAP+/− and GCAP−/− retinas without transgene, the second row (B, E, H) retinas from G1T3+GCAP+/+, G1T3+GCAP+/− and G1T3+ GCAP−/−, and the third row (C, F, I) from the G1T4+GCAP+/+, G1T4+GCAP+/− and G1T4+GCAP−/− retinas. Bar = 20 μm for all figure parts. Retinal layers are identified as OS (outer segments), IS (inner segments), ONL (outer nuclear layer containing photoreceptor nuclei) and OPL (outer plexiform layer). Download figure Download PowerPoint Figure 4.Rod response families in different mouse lines. Panels show superimposed mean responses (n ≥ 10), recorded from either GCAP+/+ (A), GCAP−/− (B) or G1T4+GCAP−/− (C–F) rods, to 10 ms flashes that increase in intensity in ∼2-fold steps from starting values (equivalent 500 nm photons/μm2/flash) of: 0.6, 2.0, 8.4, 2.0, 8.4 and 8.4, respectively. In all panels, responses were normalized to the rod's resting dark current (R/Rmax), which was: (A) −21.8 pA, (B) −13.6 pA, (C) −13.1 pA, (D) −12.8 pA, (E) −21.4 pA and (F) −11.3 pA. All flashes were delivered at 0.1 s. Download figure Download PowerPoint Figure 5.Flash response properties. Collected results were plotted for each recorded rod half-saturating flash intensity (I0.5 in equivalent 500 nm photons/μm2/flash), dim flash response integration time (response area/peak amplitude), and time-to-peak for GCAP+/+ (open circles), GCAP−/− (filled circles), G1T4+GCAP−/− (filled triangles), G1T3+GCAP−/− (open triangles) and G1T4+GCAP+/+ (filled squares). Download figure Download PowerPoint Figure 6.Paired flash ERGs. (A) Left, paired flash response of different lines of transgenic mice at 1000 ms. A conditioning flash produced 5500 isomerizations/rod and a probe flash produced 350 000 isomerizations/rod. Each trace represents the average of three mice. Right, response to the probe flash alone. (B) Graphical representation of normalized a-wave recovery of the probe flash for varying times after the test flash. Each point represents the average for 4–10 mice. Data were fit to a single exponential with the form: e[;−(t−T)/tau];. GCAP+/+ mice: T = 481 ms, tau = 481; G1T4+GCAP−/− mice: T = 511 ms, tau = 567 ms; G1T3+GCAP−/− mice: T = 722 ms, tau = 1102 ms; GCAP−/−: T = 511 ms, tau = 2476 ms. The response of each probe flash alone has been normalized to 1, and other traces are scaled accordingly. Download figure Download PowerPoint G1T4+GCAP+/+ mice, which express ∼30–50% more GCAP1 than wild type (Figure 2B), exhibit a loss of two to three photoreceptor cell layers by 4 weeks of age (Figure 3C), but do not exhibit any further degeneration even as late as 6 months of age (Baehr et al., 2000; and data not shown). The number of cone photoreceptors at 6 months of age appears to be normal. The degenerative effect in some rods is presumably due to overexpression of GCAP1, since G1T4+ mice on heterozygous and GCAP−/− backgrounds (with reduced wild-type GCAP1 levels) showed normal outer nuclear layer (ONL) thickness at advanced ages. Photoreceptor degeneration due to overexpression has commonly been seen in transgenic rhodopsin lines (Olsson et al., 1992). The lower G1T3 transgene expression level does not appear to exhibit an additive deleterious affect on the ONL in G1T3+ mice on a GCAP+/+ background. Interestingly, the G1T3+ transgenic line exhibits enhanced cone GCAP1 expression, while rod expression is greatly reduced (Figure 3H). Unlike the immunostaining seen in wild-type retina cone cells, GCAP1 in the G1T3+GCAP−/− retina is localized intensely throughout cone cells. The results seen with the G1T3+ line indicate that regulatory elements directing expression to rod photoreceptors or rod-specific enhancer elements may be located in a region −1337 to −3201 bp upstream of the translation start site. This region revealed numerous repetitive elements, including direct repeats, palindromes and developmentally regulated retinoic acid repeats (DR) (Sam et al., 1996). This region therefore is highly susceptible to structural folding, which may have contributed to rearrangement and subsequent deletion of this area during transgene insertion in the G1T3+ founder. Two DR repeats lie at positions −2244 to −2036 bp and −1883 to −1803 bp upstream of the ATG start site (Figure 1C, a and b). DR expression has been shown to be induced by retinoic acid in cultured ES cells and is expressed with tissue specificity in embryos as well as the adult mouse brain. Interestingly, between these two repetitive regions, on opposite strands, lie overlapping consensus binding sites for retinoic acid and AP1 enhancers (Figure 1C, c and d, respectively). Although little is understood concerning rod versus cone specificity in gene expression, there is evidence that retinoic acid can induce progenitor photoreceptor cells to adopt a rod cell fate (Kelley et al., 1999). Loss of this region and subsequent lack of expression of GCAP1 in rods in the G1T3+ transgenic line may have revealed a developmentally regulated program for rod expression of GCAP1 by retinoic acid. In addition, although less intense than in the G1T3+ line, the G1T4+ line also appears to show slightly higher GCAP1 staining in cone cell bodies and synaptic pedicles. This line, however, does not show a promoter deletion. It is conceivable that the transgene containing the 5.4 kb upstream regulatory sequence does not contain the necessary 'fine-tuning' regulatory elements, which may be located further upstream and contribute to the lower levels of expression in cones. Rescue of rod function by GCAP1 Light responses of rods from GCAP−/− mice are markedly slower than those of GCAP+/+ rods. Overexpression of bovine GCAP2 on a GCAP−/− background restored normal rod responses to saturating flashes but failed to restore the normal recovery kinetics to responses evoked by subsaturating flashes (Mendez et al., 2001). We used two recording techniques to determine whether GCAP1 in the absence of GCAP2 could rescue normal rod function. Responses of single rods to dim and moderate strength flashes were recorded using suction electrodes. Responses to bright flashes were evaluated by recording the ERG responses to paired flashes. Together these functional assays indicated that GCAP1 alone could rescue normal rod behavior over a broad range of light levels. Rod single-cell recordings Suction electrodes (Yau et al., 1977) were used to record light-evoked changes in circulating dark current from the outer segments of rods from wild-type and genetically modified mice. Representative response families are shown in Figure 4. As reported previously (Mendez et al., 2001), flash responses in GCAP−/− rods were more sensitive to light and slower to reach peak and recover than responses in wild-type rods (Figure 4A and B). To compare rods from mice of different genotypes, three features of the flash response were measured: the light intensity that evokes a half-saturating response (I0.5), and the time-to-peak (tpeak) and integration time (tinteg) of the linear range response (see Table I). The three response parameters were determined for recorded rods and are plotted against each other in Figure 5. In the three panels, data from wild-type (open circles) and GCAP−/− rods (filled circles) fall into separate clusters. Compared with wild-type cells, GCAP−/− rods generate flash responses that take on average about three times longer to reach peak, are nearly four times more sensitive to light and have 2- to 3-fold longer integration times (Table I). In contrast, the three properties of the flash responses recorded from the G1T4+GCAP−/− rods show large rod-to-rod variations. In some rods the responses are indistinguishable from wild-type while in others they are slower, with properties between those of wild-type and GCAP−/− rods. This is apparent in Figure 4C–F, where response families have been selected from G1T4+GCAP−/− rods to illustrate the range of rod-to-rod variation in the kinetics of the responses. Table 1. Flash response properties for all recorded cells Rod type Integ. time (ms) ± SEM Range (low–high) Time to peak (ms) ± SEM Range (low–high) Intensity I0.5 ± SEM Range (low–high) Imax (pA) ± SEM Range (low–high) No. of cells Wild type 313 ± 20 192–472 206 ± 11 157–320 29.5 ± 1.5 21.5–41.9 −14 ± 0.9 −6.4 to −21.8 17 GCAP−/− 759 ± 32 624–1009 609 ± 20 481–731 6.9 ± 0.4 4.8–10.2 −12.7 ± 0.4 −9 to −14.7 13 G1T4+GCAP−/− (all cells) 410 ± 33 121–654 284 ± 26 164–542 24.5 ± 2.2 9.4–40.4 −15 ± 0.7 −10.1 to −21.4 22 G1T3+GCAP−/− 694 ± 65 567–970 513 ± 25 483–618 6.7 ± 0.7 4.5–8.3 −12.1 ± 0.4 9.1 to −14.7 6 G1T4+GCAP+/+ 408 ± 24 339–607 245 ± 23 188–411 26.9 ± 1.8 16.1–35.2 −14.4 ± 1.1 −10.7 to −22.7 10 G1T4+GCAP−/− with integ. time 472 ms 578 ± 17 518–653 402 ± 31 246–542 13.1 ± 1.3 9.4–19.3 −14.9 ± 1.3 −10.1 to −21.3 8 The mean ± SEM and range of individual measures are reported for each rod type. Integ. time, integration time (response area/peak amplitude); Imax, maximal current [pA]. Measurements of the three standard response parameters for all the recordings from G1T4+GCAP−/− rods are plotted in Figure 5 (filled triangles). The results from the different rods do not form a single cluster of data points. The data points intersperse with wild-type, but also extend into part of the range of the results from GCAP−/− rods. This observation is consistent with variable expression of GCAP1 in the G1T4+GCAP−/− rods (see below). In the bottom rows of Table I, G1T4+GCAP−/− rods are divided into two groups based on whether the integration time of the linear range response is less than or greater than the longest wild-type integration time (472 ms). Fourteen of 22 G1T4+GCAP−/− rods that fall into the group with shorter integration times and have mean values of I0.5, tpeak and tinteg that do not differ significantly from wild type (Table I). This indicates that despite rod-to-rod variation, the majority of G1T4+GCAP−/− rods generate flash responses with wild-type properties. Flash responses of rods from the G1T3+ line (G1T
Referência(s)