Rec−/− Retinal Morphology and Protein Content
The targeting vector disrupted the first exon of the recoverin gene and prevented its expression (). The effect of recoverin knockout on gene expression in the retina was analyzed using Affymetrix Murine genome U74Av2 arrays, which contain all known phototransduction genes in addition to ~12,000 functionally characterized genes and expressed sequence tags. Comparisons of expression profiles between dark-adapted rec−/− and control retinas revealed only two significant changes. There was a 10-fold decrease in the signal for recoverin. The persistence of a low signal for recoverin in knockouts could result from the presence of a partial mRNA sequence for recoverin or from weak crosshybridization with transcript for another protein. In addition, two probe sets for transducin β-subunit mRNA revealed 6- and 16-fold decreases, respectively. This large difference in expression levels for transducin β was verified using semiquantitative, real time RT-PCR. The lack of immunohistochemical staining of photoreceptor and bipolar cells in retinal sections and the absence of recoverin in Western blots of retinal protein confirmed that recoverin was indeed absent from knockout mice. Western analyses, however, did not show large changes in the amount of transducin β-subunit. Levels of rhodopsin, rhodopsin kinase, arrestin, transducin α-subunit, the α-, β-, and γ-subunits of PDE, RGS9-1, and guanylate cyclase E were also similar in knockout and control retinas. Disruption of recoverin expression was therefore considered to be functionally selective in that it did not give rise to compensatory changes in the levels of phototransduction proteins or to changes in the levels of mRNA for proteins not directly involved in phototransduction.
Knockout of recoverin did not appear to adversely affect the development or long-term viability of the retina. Rods elaborated outer segments of normal size and the outer nuclear layer, consisting of photoreceptor nuclei, attained normal thickness. The thickness of the inner nuclear layer, which contains the somas of bipolar cells, was also normal. There was no sign of a retinal degeneration in rec−/− mice up to 1 yr of age, the oldest examined.
Faster Flash Response Recovery in Dark-adapted rec−/− Rods
A shows flash response families from a representative rec−/− and control rod. Although the rising phases of the responses were similar, rec−/− responses recovered more rapidly—a characteristic difference that is analyzed more fully below. After flashes of intermediate strength, the recovery phase of the knockout rod response often displayed a prominent “kink” usually not present in responses of control rods. The kink in the flash response of rec−/− rods may explain the anomalous electroretinograms of rec−/− mice, which exhibit two corneal positive b-waves rather than one in response to a single midrange scotopic flash (Ron Bush, personal communication; Hurley and Chen, 2001
Collected results on the relation between normalized peak response amplitude and normalized flash strength are shown in B. The smooth curve, a saturating exponential (Lamb et al., 1981
), provided a good fit to the results from both populations. Although the half-saturating flash strength was lower for rec−/− rods, the difference was small, as shown in . The table also summarizes other parameters of the flash responses from control and rec−/− rods. It can be noted that the dark current and the time-to-peak of the dim flash response were very similar for the two populations, as was the estimated amplitude of the single photon response.
Response Parameters of Control and Rec−/− Rods
C compares the responses of the rods in A to flashes whose strength varied over a wide range. For subsaturating flashes, the responses of the rec−/− rod recovered more rapidly than those of the control rod. The integration time of the dim flash response, defined as the area under the response divided by its peak height, was on average 37% lower in rec−/− rods than in control rods (). The difference in effective response duration became more pronounced at higher flash strengths that saturated the response (, see also Hurley and Chen, 2001
The diminished time in saturation (Tsat) of rec−/− responses to bright flashes corresponds to an effective decrease in phototransduction gain. The magnitude of the decrease was assessed from the ratio of the flash strengths required to hold control (n = 20) and rec−/− (n = 11) rods in saturation for a given time (e.g., ). Although the slope of the relation for the two types of rod differed slightly, a flash approximately ninefold brighter was required to hold knockout rods in saturation, indicating a ninefold lower gain ( B). The general conclusion from these experiments is that wild-type rods had a higher transduction gain during the recovery phase of the dark-adapted flash response.
Effect of Background Light on PDE Activity Evoked by a Bright Flash
Early biochemical studies suggested that recoverin exerts its effect in the Ca2+
-bound form (Kawamura, 1993
). If Ca2+
-bound recoverin prolongs the response to a bright flash delivered in darkness, then at low Ca2+
, control and rec−/− rods should behave identically. This idea was tested using the step-flash protocol devised by Fain et al. (1989)
, as shown in A. Steady background lights of varying intensities were used to lower the rod's [Ca2+
, before the presentation of a saturating test flash. As expected (Fain et al., 1989
), background light shortened the saturation time of the response to the flash in control rods (). Because the test flash response itself always lowered [Ca2+
to a minimal level and thus activated guanylate cyclase maximally, the reduction in Tsat
should be caused by a reduction in the flash-evoked PDE activity (Matthews, 1996
). The shortening of Tsat
was a robust phenomenon, being present in each of 34 control cells.
Background light failed to shorten the Tsat of rec−/− rods (19 of 20 rods) and in most cases increased it. Records from one such experiment are shown in C. Tsat was extended slightly with conditioning step intensity in rec−/− rods because recovery from the step alone was generally more prolonged at higher intensities. There was some evidence that the same effect partially masked the decline in Tsat with conditioning step intensity for control rods. In 7 out of 24 control rods where a wide range of conditioning step intensities were tested, Tsat change little or even increased after dim to moderate conditioning steps, but eventually decreased at higher step intensities. Background light did shorten Tsat slightly in one rec−/− rod. But the recovery phases of the responses to steps and steps plus flashes in this rod had undershoots that were not present in the responses to the flashes alone. Similar behavior was sometimes observed in control rods and hence appears to be unrelated to the deletion of recoverin. The fact that background light caused the saturation time of the response to a bright test flash in control rods and rec−/− rods to become more similar (, B–D) is consistent with the notion that Ca2+-recoverin boosts the latter portion of the bright flash response.
Altered Kinetics of Na+/Ca2+-K+ Exchange in rec−/− Rods
The light response is generated by changes in the concentration of cGMP, which in turn reflect not only activation of PDE by light but also Ca2+
mediated activation of guanylate cyclase by GCAPs (guanylate cyclase activating proteins). Recoverin's abundance in photoreceptors (17–40 μM; Kawamura and Murakami, 1991
; Klenchin et al., 1995
), suggests that it could contribute significantly to a rod's Ca2+
buffering capacity. Thus, the deletion of recoverin could speed the light-induced decline in calcium and the resulting activation of guanylate cyclase. To assess the effect of recoverin deletion on Ca2+
dynamics, we recorded the Na+
exchange currents of control and knockout rods. A bright flash, which rapidly closed the light-sensitive channels, revealed the exchange current as a small, slowly decaying inward current (). The exchange transients are shown on an expanded, normalized ordinate scale in the lower panels (jagged traces) where they are fitted with single exponentials (smooth curves). In control rods the exponential time constant was 113 ± 7 ms (mean ± SEM), while in rec−/− rods, it was 86 ± 9 ms (). In the same rods the initial amplitude of the exchange current, extrapolated to the time at which the bright flash response reached half its peak amplitude (Yau and Nakatani, 1985
), was −0.53 ± 0.02 and −0.59 ± 0.05 pA in control and rec−/− rods, respectively. Because the stoichiometry of ionic exchange is fixed (Cervetto et al., 1989
) the time integral of the exchange current is directly proportional to the amount of Ca2+
extruded. The initial amplitudes were the same for the two types of rods, consistent with their maintaining a similar free [Ca2+
in darkness. However, the longer time constant for control rods made the integrated area of the exchange current larger, indicating that more Ca2+
was extruded from control rods. The 9.1 fC of additional charge entering control rods during the exchange current corresponds to 6 to 10 μM Ca2+
in the outer segment, assuming a volume range of 9.2 and 15.4 fl (see materials and methods
). Since most of a rod's internal Ca2+
is bound (Lagnado et al., 1992
), the extra Ca2+
content of controls presumably represents a fraction bound to recoverin. This fraction, which is apparently released and extruded rapidly after a bright flash, comprises ~15% of the total Ca2+
present in the outer segment.
Figure 5. Accelerated Ca2+ dynamics in rec−/− rods. The top panel shows the averaged responses of a control and a knockout rod to bright, saturating flashes of 1,690 and 5,520 photons μm−2, respectively. The response amplitude was (more ...)
Does the rec−/− phenotype result simply from a faster fall in intracellular Ca2+ concentration and earlier activation of guanylate cyclase? We examined this in two ways.
First, we used BAPTA-AM incorporation to bolster the Ca2+ buffering capacity and slow the changes in intracellular free Ca2+ concentration in four rec−/− rods from one retina. Ca2+ buffering was significantly increased as demonstrated by the fact that the exchange current time constant was twofold longer than that in four untreated rods from the other retina of the same mouse (82 ± 13 ms, rec−/−; 157 ± 29 ms, rec−/− with BAPTA). BAPTA did not restore a normal phenotype to rec−/− rods; instead it delayed the time to peak of the flash response and introduced an oscillation in the recovery phase ( E, inset). Furthermore, background light still failed to reduce the saturation time of a bright flash response ( E). These experiments suggest that recoverin does not act only as a Ca2+ buffer in the outer segment.
Second, we determined the effect of deleting recoverin in rods lacking calcium-regulated GC activity. We did so by comparing transduction in rods lacking guanylate cyclase–activating proteins (GCAPs−/−; Mendez et al., 2001
) with that in rods lacking both GCAPs and recoverin (GCAPs−/− rec−/− double knockouts). If recoverin solely acts by controlling the time course of cyclase activation, deleting recoverin should have no effect in the GCAPS−/− background. illustrates single photon responses averaged from a number of GCAPs−/−, GCAPs−/− rec−/− double knockouts, and wild-type rods; summarizes several parameters of the flash responses of the cell populations. As reported previously, GCAPs deletion increased the amplitude, time-to-peak, and integration time of the single photon response (Mendez et al., 2001
). Deletion of recoverin in the GCAPs−/− background gave a sizeable reduction in the single photon response amplitude (, ) and a corresponding increase in the half-saturating flash strength (). Recoverin deletion also shortened the time-to-peak and integration time of the dim flash response in the GCAPs−/− background. This indicates that in the absence of recoverin, light-stimulated PDE activity was effectively shorter. In addition, the “kinks” observed in subsaturating rec−/− responses were absent in the GCAPs−/− rec−/− responses. Apparently, the kinks in the responses of the rec−/− rods can be attributed to abnormally rapid cyclase activation. The characteristic differences in the flash responses of normal, rec−/−, GCAPs−/−, and GCAPs−/− rec−/− were satisfactorily predicted by a simple quantitative model of the response dynamics ( and ).
Figure 6. Changes in the flash responses of GCAPs−/− rods after deletion of recoverin. (A) Single photon responses of GCAPs−/− and GCAPs−/− rec−/− rods. Traces show the averaged responses of 15 rods (more ...)
Flash Response Parameters of Rods after Deletion of GCAPs and Recoverin
Figure 8. Calculated flash responses. (A) Assumed time courses of rhodopsin's catalytic activity. In the control rod (top), activity shut off exponentially after a delay of 100 ms (Chen et al., 1995b, 1999). The time constant of 205 ms was found from the mean slope (more ...)
Saturation times of bright flash responses in GCAPs−/− rods were shifted to twofold higher flash strengths upon recoverin deletion (e.g., B; n = 21 GCAPs−/− and 11 GCAPs−/− rec−/− rods). In additional experiments (not depicted), exposure to a conditioning step shortened the saturation time of the response to a bright flash in each of 11 GCAPs−/− rods, as expected from the presence of recoverin's action, but this effect was absent in each of 7 GCAPs−/− rec−/− rods.
In summary, these results suggest that recoverin has a dual action on the flash response: (a) Ca2+-recoverin extends the effective duration of rhodopsin's catalytic lifetime, and (b) by acting as a Ca2+ buffer, recoverin slightly delays the activation of guanylate cyclase.
Decreased Sensitivity of rec−/− Rods to Steps of Light
The onset of a step of light elicited a response that rose to a peak and then partially recovered, as the rod adapted (e.g., A). The initial restoration of the current was faster in rec−/− rods than in controls ( A, arrowheads), but was not faster in rods lacking both GCAPs and recoverin (GCAPs−/− rec−/−; unpublished data). This suggests that the initial, faster restoration of the current in rec−/− rods arises from accelerated Ca2+ activation of guanylate cyclase. The absence of recoverin decreased step sensitivity, measured at the initial peak of the step response ( B and ). For control rods, an intensity of 250 photons μm−2 s−1 elicited a half-maximal response while for rec−/− rods, 410 photons μm−2 s−1 was required (). This 1.6-fold difference is attributable to the 1.6-fold lower integration time of the dim flash response in rec−/− rods.
Incremental Flash Responses in Background Light
Deleting recoverin had little effect on light adaptation, as assessed by measuring the dependence of flash sensitivity on background light intensity ( C). The plot shows the relative sensitivity to a dim test flash as a function of normalized background light intensity, with both scales logarithmic. The closed symbols show results from control rods, and open symbols show results from rec−/− rods. The normalizing factor for the background light intensity is I0
, the intensity that reduced the cell's flash sensitivity to half the value in darkness. Values of I0
were similar in control and rec−/− rods: 178 and 192 photons μm−2
, respectively ().
The smooth curve is drawn according to the Weber-Fechner relation:
This expression provides an empirical description of the behavior observed in several types of mammalian rods (Tamura et al., 1989
; Nakatani et al., 1991
). For comparison, the dashed and dotted curves plot the relation expected for rods whose adaptation results strictly from response saturation:
The behavior of both types of rod was very similar, and both populations truly adapted rather than simply saturating. We conclude that deletion of recoverin had little effect on the mechanisms that fix the dependence of incremental flash sensitivity on background light intensity.