Receptor demise from alteration of glycosylation site in Drosophila opsin: Electrophysiology, microspectrophotometry, and electron microscopy

In the δAsn20 Drosophila stock, the N-linked glycosylation site of opsin in Rl-6 receptors (Rhl) is absent. We used electroretinography (ERG), microspectrophotometry (MSP), and electron microscopy (EM) to quantify visual cell defects. Positive controls, w9, had wild type Rhl. MSP revealed minimal photopigment in δAsn20 for 6 days posteclosion; w9 had near normal visual pigment. ERG sensitivity and prolonged depolarizing afterpotential (PDA) were compared for δAsn20 and w9. δAsn20's Rl-6 function is decreased 100–fold at eclosion and diminishes until only R7/8 functions at 11 days. What little rhodopsin is routed to the rhabdomere functions. Morphometry showed smaller Rl-6 rhabdomeres in δAsn20 for 8 days posteclosion. Rhabdomeres in w9 were normal. A negative control, ninaE0117, a deletion of the Rhl gene, also has small rhabdomeres. δAsn20 and ninaE0117 lack the extreme rhabdomere elimination of ora (outer rhabdomeres absent), a nonsense mutant interrupting Rhl's coding sequence. δAsn20 and ora have surplus membrane while ninaE0117 does not. Freeze fracture reveals that δAsn20's rhabdomeric P-face particle count is as low as for vitamin A deprivation, consistent with an opsin defect. High particle density, organized into rows, is present in adjacent plasmalemma where surplus membrane accumulates. In summary, δAsn20 interferes with either synthesis, deployment, or maintenance of opsin.

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. I. The induction process.

In invertebrate photoreceptors, when the light stimulus results in substantial net transfer of the visual pigment from the rhodopsin (R) to the metarhodopsin (M) state, the ordinary late receptor potential (LRP) is followed by a prolonged depolarizing afterpotential (PDA). The dependence of the amplitude of the PDA on the amount of pigment conversion is strongly supralinear, and the PDA duration also depends on this amount. These observations indicate an interaction among the elements of the PDA induction process and also make possible a test of the range of this interaction. The test consists of a comparison of the PDA after localized pigment conversion, obtained by strong spot illumination, to that after weaker diffuse illumination converting a comparable total amount of pigment. The experiment was performed on the barnacle lateral eye. The effective spot size was measured by the early receptor potential (ERP), in seawater saturated with CO2, which considerably reduced the electrical coupling between the photoreceptors. The ERP was also used to determine whether there is diffusion of R molecules into the illuminated spot. The spot illumination induced a PDA with small amplitude and long duration, while no detectable PDA was induced by the diffuse light. This indicates that the range of the PDA interaction is much smaller than the entire cell. In addition, the ERP results showed that there was no detectable diffusion of R molecules into the illuminated spot area over 30 min. This measurement, with a calculated correction for the microvillar geometry of the photoreceptor, enabled us to put an upper limit on the diffusion coefficient of the pigment molecules in the inact, unfixed barnacle photoreceptor of D less than 6 X 10(-9) cm2 s-1.

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. II. Antagonistic interactions.

In the preceding article, we investigated the spatial properties of the induction of the prolonged depolarizing afterpotential (PDA) by shifting visual pigment from the rhodopsin (R) to the metarhodopsin (M) state in the barnacle photoreceptor. In this work, we have studied the ranges within the cell of the antagonistic effects on the PDA of M-to-R transfer. When this transfer occurs during a PDA, it depresses the PDA; when it precedes PDA induction, it impedes that induction ("anti-PDA"). These ranges were previously shown (by a statistical technique) to be at least a few tens of nanometers within a half-second (D greater than 10(-13) cm2 s-1). We now demonstrate, with local illumination techniques in which a PDA was induced in one side of the cell and PDA depression or anti-PDA was induced in the other side, that both ranges are much smaller than the cell diameter (approximately 100 microns) within 30 s (D less than 10(-6)). We further show, using a less direct but shorter-range technique involving colored polarized light, that the interaction of the PDA with the anti-PDA is restricted to less than approximately 6 microns (D less than 6 X 10(-9)). This figure is quite low and suggests that the interaction may be confined to the pigment molecules, possibly in a complex of the type suggested in the preceding article.

Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster.

A Drosophila mutant (ninaAP228) that is low in rhodopsin concentration but identical to the wild-type fly in photoreceptor morphology has been isolated. R1-6 photoreceptors of the mutant differ from those of wild type in that (a) the prolonged depolarizing afterpotential (PDA) is absent, (b) concentrations of rhodopsin and opsin are substantially reduced, and (c) intramembrane particle density in the membranes of the rhabdomeres is low. Each of these traits is mimicked by depriving wild-type flies of vitamin A. The ninaAP228 mutation differs from vitamin A deprivation in that in the mutant (a) the rhabdomeric membrane particle density is reduced only in the R1-6 photoreceptors and not in R7 or R8, (b) the PDA can be elicited from the R7 photoreceptors, and (c) photoconversion of R1-6 rhodopsin to metarhodopsin by ultraviolet (UV) light is considerably more efficient than in vitamin A-deprived flies. The absorption properties of the mutant rhodopsin in the R1-6 photoreceptors appear to be identical to those of wild type as judged from rhodopsin difference spectra. The results suggest that the mutation affects the opsin, rather than the chromophore, component of rhodopsin molecules in the R1-6 photoreceptors. The interaction between the chromophore and R1-6 opsin, however, appears to be normal.

The distribution of bumps in the tail of the locust photoreceptor afterpotential

An extended tail or prolonged depolarizing afterpotential (PDA) follows the receptor potential of a locust retinula cell when the stimulating light is in the intensity range that saturates the receptor potential. The amplitude and duration of this afterpotential depend on the intensity and duration of the stimulus. As the afterpotential decays, apparently exponentially, it becomes resolved into bumps, which we call light-induced dark bumps (LID bumps). The intervals between light-induced dark bumps are distributed in a way that is indistinguishable from a random (Poisson) distribution. As previously demonstrated, LID bumps are indistinguishable from bumps directly induced by low intensity light in light-adapted cells, which in turn grade into the slightly larger bumps produced, each by a single photon, in dark-adapted cells. The light-induced dark bumps continue for up to an hour in darkness, slowly becoming like dark-adapted bumps in amplitude and shape. To account for the random occurrence and discrete features of bumps after so long a latency, we propose that intense light generates a significant amount of an intermediate molecule or packet which decays slowly to start the same process that normally generates bumps with a short delay.