Abstract
RGS9 and R9AP are components of the photoreceptor-specific GTPase activating complex responsible for rapid inactivation of the G protein, transducin, in the course of photoresponse recovery from excitation. The amount of this complex in photoreceptors is strictly dependent on the expression level of R9AP; consequently, the knockouts of either RGS9 or R9AP cause comparable delays in photoresponse recovery. While RGS9 is believed to be present only in rods and cones, R9AP is also expressed in dendritic tips of ON-bipolar cells, which receive synaptic inputs from photoreceptors. Recent studies demonstrated that knockouts of R9AP and its binding partner in ON-bipolar cells, RGS11, cause a small delay in ON-bipolar cell light responses manifested as a delayed onset of electroretinography b-waves. This led the authors to suggest that R9AP and RGS11 participate in regulating the kinetics of light responses in these cells. Here we report the surprising finding that a nearly identical b-wave delay is observed in RGS9 knockout mice. Given the exclusive localization of RGS9 in photoreceptors, this result argues for a presynaptic origin of the b-wave delay in this case and perhaps in the case of the R9AP knockout as well, since R9AP is expressed in both photoreceptors and ON-bipolar cells. We also conducted a detailed analysis of the b-wave rising phase kinetics in both knockout animal types and found that, despite a delayed b-wave onset, the slope of the light response is unaffected or increased, dependent on the light stimulus intensity. This result is inconsistent with a slowdown of response propagation in ON-bipolar cells caused by the R9AP knockout, further arguing against the postsynaptic nature of the delayed b-wave phenotype in RGS9 and R9AP knockout mice.
Highlights
At the first step of visual processing in the vertebrate retina, photoreceptors convey light-evoked signals to bipolar cells [1]
R9AP expression in ON-bipolar cell dendrites and photoreceptor synaptic morphology are not affected by the RGS9 knockout The goal of our study was to conduct a comprehensive comparison of ERG responses in R9AP2/2 and RGS92/2 mice
This required two control experiments addressing: (1) whether the expression of R9AP in dendritic tips of ON-bipolar cells is affected by the RGS9 knockout, and (2) whether either R9AP or RGS9 knockout affects the morphology of the photoreceptor-to-ONbipolar cell synapses
Summary
At the first step of visual processing in the vertebrate retina, photoreceptors convey light-evoked signals to bipolar cells [1]. Both cell types rely on G protein-mediated signaling pathways for generating their light-responses. While transducin has the intrinsic ability to hydrolyze bound GTP, the rate of this reaction is slow and, in photoreceptors, it is accelerated roughly 100-fold by the GTPase activating protein, RGS9 [6,7,8]. In addition to tethering RGS9?Gb5 on the surface of photoreceptor discs, R9AP enhances the ability of RGS9?Gb5 to activate transducin GTPase [11,12], directs RGS9?Gb5 to outer segments [13,14], and protects RGS9?Gb5 from intracellular proteolysis, setting the expression level of the entire RGS9?Gb5?R9AP complex. The protective role of R9AP was established by demonstrating that R9AP knockout causes complete elimination of RGS9 from photoreceptors [15], whereas R9AP overexpression in rods increases the amounts of RGS9 and Gb5 as well [16]
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