Optoretinography in R9AP-Bradyopsia Reveals an Essential role of G-protein Activation and Deactivation in the Human Cone Elongation Response

Abstract

Photoreceptor outer segments (OS) shorten and elongate in response to bleaching stimuli: these responses are measurable optically at nanometer scale resolution with OCT and collectively known as the optoretinogram (ORG). The molecular mechanisms underlying the ORG remain unresolved. Here we examine the role of G-protein in cone elongation by measuring ORGs in 2 female bradyopsia subjects caused by mutation in R9AP, the membrane anchor protein of RGS9, necessary for the latter’s transport to the OS and for normal deactivation of phototransduction by the GTPase-activating protein (GAP) complex. The ORGs of bradyopsia subjects had normal activation kinetics, amplitude and photosensitivity, but markedly slowed recovery compared to controls (2 males, 1 female). The slowed recovery is attributable to a reduced level of RGS9 in the OS and localizes the molecular basis of the OS elongation in the G-protein cascade sequence at, or prior to, formation of the GAP complex. The recovery of the ORG, measured in a paired-flash paradigm, revealed that the elongation requires a substrate that is depleted and recovers in a bleach level-dependent manner. Recovery from the highest bleach (75%) tracked the time course of cone opsin regeneration, implying that unregenerated cone opsin produces “ dark light ”, known to arise in rods from activation of G-protein by unregenerated rhodopsin. Overall, our results identify holo-trimeric G-protein as an essential and rapidly recyclable substrate for the ORG elongation response, and establish the ORG as a non-invasive, molecularly interpretable optical assay of G-protein signaling and dark adaptation suitable for evaluating diseases and therapies. Significance statement Photoreceptor outer segments shorten and elongate in response to bleaching stimuli: these responses are measurable optically at nanometer scale resolution with OCT and known as the optoretinogram (ORG). The molecular mechanisms underlying the ORG remain unresolved. By comparing control ORGs against subjects with a mutation in the anchor protein of regulator of G-protein signaling RGS9, we identified G-protein as an essential and rapidly recyclable substrate for the ORG elongation response. The recovery from exposures isomerizing 75% opsins tracked the time-course of pigment regeneration, indicating persistent activation of G-protein by unregenerated opsin. By linking ORG dynamics to defined molecular reactions, this work establishes the ORG as a quantitative, noninvasive assay of G-protein signaling and cone dark adaptation suitable for clinical translation.