Human cone elongation responses can be explained by photoactivated cone opsin and membrane swelling and osmotic response to phosphate produced by RGS9-catalyzed GTPase

Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (∼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (∼425 nm) and initial rate (∼3 nm ms⁻¹) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein–phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.

Cone photoreceptors initiate the high-acuity daytime vision of healthy humans. The loss of central retinal cone function, as occurs in age-related macular degeneration, is personally devastating and medically extremely costly (1, 2). Over the past 25 y, thanks to the development of optical coherence tomography (OCT) (3, 4) and the application of adaptive optics (AO) to retinoscopy (5–7), there have been enormous advances in imaging the cone mosaic at single-cell resolution in the living eye. Very recently, nanometer-scale measurements of the optical path length (OPL) of rod and cone outer segments (COSs) have made it possible to “optoretinographically” quantify light-driven length changes of individual human COSs (8–14), affording a cell by cell assessment of normal cone function and of dysfunction in disease states (15), with great potential value for evaluating therapeutic interventions. Other than the dependence of rod outer segment (ROS) elongation on the G protein transducin (16), no specific molecular mechanisms of light-driven outer segment elongation, however, have been identified.In this investigation, hypotheses for the molecular mechanisms of human COS elongation are proposed and tested. The kinetics and bleaching sensitivity of the COS responses were measured in response to brief light stimuli ranging 200-fold in intensity, up to and including intensities that bleach the entire complement of cone opsin. The bleach-level dependence and kinetics strongly constrain hypotheses that could underlie COS elongation, rejecting G-protein subunits dissociated from the membrane, by-products of phosphodiesterase-catalyzed cyclic guanosine monophosphate (cGMP) hydrolysis or of guanylate cyclase synthesis of cGMP, and changes in ionic concentrations. Application of a model of cone phototransduction appropriate for the extreme bleaching levels involved supports the hypotheses that COS elongation is partly driven by the osmotic response to free phosphate (P_i) produced by the regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in the G_tcα–phosphodiesterase (PDE) complex and partly driven by bleaching-induced swelling of cone opsin (17) and disk membrane expansion attendant chromophore hydrolysis and its reduction to all-trans retinol (at-ROL) (18).