Identification of key factors that reduce the variability of the single photon response

Abstract

Rod photoreceptors mediate vision in dim light. Their biological function is to discriminate between distinct, very low levels of illumination, i.e., they serve as reliable photon counters. This role requires high reproducibility of the response to a particular number of photons. Indeed, single photon responses demonstrate unexpected low variability, despite the stochastic nature of the individual steps in the transduction cascade. We analyzed individual system mechanisms to identify their contribution to variability suppression. These include: (i) cooperativity of the regulation of the second messengers; (ii) diffusion of cGMP and Ca 2þ in the cytoplasm; and (iii) the effect of highly localized cGMP hydrolysis by activated phosphodiesterase resulting in local saturation. We find that (i) the nonlinear relationships between second messengers and current at the plasma membrane, and the cGMP hydrolysis saturation effects, play a major role in stabilizing the system; (ii) the presence of a physical space where the second messengers move by Brownian motion contributes to stabilization of the photoresponse; and (iii) keeping Ca 2þ at its dark level has only a minor effect on the variability of the system. The effects of diffusion, nonlinearity, and saturation synergize in reducing variability, supporting the notion that the observed high fidelity of the photoresponse is the result of global system function of phototransduction.modeling | rhodopsin | deactivation | phosphorylation I n vertebrate retinal rod photoreceptors, light-activated rhodopsin R Ã activates dozens of G proteins (T → T Ã ) during its random lifetime, by catalyzing GDP/GTP exchange on the G protein α-subunit. Each T Ã molecule associates, one-to-one, with a catalytic subunit of the effector, forming an active T Ã -E complex, denoted by E Ã . Molecules of E Ã hydrolyze the second messenger, cGMP. Reduction of the cGMP level induces its dissociation from the cGMP-gated cation channels, causing channel closure and suppression of the inward current. This current suppression is the experimentally measured electrophysiological response, which is usually normalized by the dark current j dark , yielding the relative current suppression (1)The mechanism of R Ã deactivation contains several random components, including the random number of phosphorylations by rhodopsin kinase (RK), and the random sojourn time at each phosphorylation level. These steps regulate the number of generated E Ã molecules and, hence, the response. Because of this randomness the responses are expected to be inherently variable, in the sense that any two rhodopsin photoisomerizations yield different responses. The system would be stable if these responses are statistically close. A measure of stability is the coefficient of variation (CV), defined as the ratio of the standard deviation to the mean, calculated over a large number of signaling events and their corresponding responses. However, the measured single photon response (SPR) is highly reproducible, i.e., the coefficient of var...

The phototransduction machinery in the rod outer segment has a strong efficacy gradient

“…Previous studies in the early 1980s have demonstrated a lower sensitivity and slower kinetics of flash responses (3,17,18) at the tip of the OSs and that the amplitude of the single photon response is smaller at the OS tip than at the OS base (18). Despite these experimental observations, most of the models of phototransduction, developed subsequently, assumed a homogeneous efficacy of the transduction machinery along the OS (1,(19)(20)(21)(22). In the present paper, by using highly confined spots of light obtained with apertureless tapered optical fibers (TOFs), we reveal that the biochemical machinery inside the rod OS has a gradient of efficacy much larger than previously thought (17,18), demonstrating that the assumption of a homogeneous OS is not true.…”

Sperm as microswimmers – navigation and sensing at the physical limit

“…150 ms, the GC * synthesizes about 11-45 cGMP molecules [4,17]. For comparison, in photoreceptors, which also use a cGMP-signalling pathway, a single photon stimulates the hydrolysis of 2,000 to 72,000 cGMP molecules depending on the species [54,55]. Thus, at this stage of signalling, the amplification in photoreceptors is several orders of magnitude larger.…”

“…For rhodopsin, the visual pigment in photoreceptors, stepwise inactivation by two phosphorylation steps and "capping" of phosphorylated rhodopsin by a "stop" protein has been proposed to control its lifetime and, thereby, reduce photon noise [49][50][51]. However, uniform single-photon responses also involve other mechanisms, including Ca 2+ feedback [52][53][54].…”

Engineering Visual Arrestin-1 with Special Functional Characteristics

“…Rods demonstrate remarkable single photon sensitivity (1) and unusual reproducibility of a single photon response (2)(3)(4). These features require biochemically perfect signaling cascade with virtually no noise, enormous amplification, and extremely rapid shutoff.…”