Early receptor potential: photoreversible charge displacement in rhodopsin

When the eye is illuminated by an intense flash, the visual pigment rhodopsin begins to pass rapidly through a series of intermediate states, eventually becoming bleached. If a second flash is delivered during the lifetimes of these intermediates the rhodopsin can be photoregenerated. A fast electrical response of the visual receptors, the early receptor potential, is elicited by the first flash. A similar response is elicited by the second flash, but the polarity of this response is reversed. Moreover, this response can be separated into three components, each arising from the action of light on a different intermediate. It is likely that all these fast responses, including the early receptor potential, arise from charge displacements in the visual-pigment molecule.     

Molecular and thermal origins of fast photoelectric effects in the squid retina

When a short, intense flash of light is absorbed by the outer segments of squid photoreceptors fixed in glutaraldehyde, a voltage appears briefly across the retina. The waveform depends on the relative amounts of rhodopsin and its various stable photoproducts present at the beginning of the flash. Each light-absorbing species present contributes a characteristic voltage component which is summed in the gross waveform. The heating effect of the absorbed light produces a small, long-lasting thermoelectric voltage as well. When this thermal effect is corrected for, interconversion of equal numbers of rhodopsin and acid metarhodopsin molecules by a flash results in a fast voltage waveform whose time integral is zero. Thus the charge flowing in one direction in the retina when rhodopsin is converted to acid metarhodopsin by one photon is apparently exactly reversed when acid metarhodopsin is reconverted to rhodopsin by another.     

Rhodopsin photoproducts: effects on electroretinogram sensitivity in isolated perfused rat retina

Isolated perfused retinas of albino rats were exposed to brief saturating flashes of white light which bleached about 50 percent of the rhodopsin present. Transient photoproducts of the reaction could be detected for about 30 minutes. The b-wave threshold increased by some 3 logarithmic units immediately after the flash and remained stable at this level thereafter. This suggests that the longer-lived intermediate products of rhodopsin photolysis do not influence scotopic visual sensitivity.     

Function of rhodopsin in temperature discrimination in Drosophila

Many animals, including the fruit fly, are sensitive to small differences in ambient temperature. The ability of Drosophila larvae to choose their ideal temperature (18°C) over other comfortable temperatures (19° to 24°C) depends on a thermosensory signaling pathway that includes a heterotrimeric guanine nucleotide-binding protein (G protein), a phospholipase C, and the transient receptor potential TRPA1 channel. We report that mutation of the gene (ninaE) encoding a classical G protein-coupled receptor (GPCR), Drosophila rhodopsin, eliminates thermotactic discrimination in the comfortable temperature range. This role for rhodopsin in thermotaxis toward 18°C was light-independent. Introduction of mouse melanopsin restored normal thermotactic behavior in ninaE mutant larvae. We propose that rhodopsins represent a class of evolutionarily conserved GPCRs that are required for initiating thermosensory signaling cascades.     

Rhodopsin mutants that bind but fail to activate transducin

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.     

Photoelectric potential from photoreceptor cells in ventral eye of Limulus

Intense colored light from a gas laser evokes a photoelectric potential in the photoreceptor cells of the ventral eye of Limulus. This potential has two components, both of which have the action spectrum of a rhodopsin with an absorbancy maximum at 530 nm. The evidence is consistent with the hypothesis that the photoelectric potential arises directly from the orderly array of rhodopsin molecules which are an integral part of the photoreceptor cell membrane.     

Corrections and clarifications

In the report "Activation and regeneration of rhodopsin in the insect visual cycle" by A. Kiselev and S. Subramaniam (25 Nov., p. 1369), the second and third sentences of the last paragraph on page 1371 were incorrectly printed. They should have read, "When rhodopsin was re-excited immediately after it was regenerated, most of the metarhodopsin formed was thermally stable (that is, M(b) was formed predominantly; Fig. 4A). However, when rhodopsin was re-examined after waiting for various lengths of time (in the dark), an exponential increase was observed in the amount of thermally unstable metarhodopsin produced, implying heterogeneity in the rhodopsin population."     

Temporal summation of light by a vertebrate visual receptor

Using aspartate to isolate mass receptor-activity, we have investigated the reciprocity of flash intensity and flash duration in determining the response of the frog's cone receptor. The duration over which reciprocity holds decrease with increases in either flash energy of ambient light intensity. These findings parallel those of human psychophysical experiments.     

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.     

Multiple phosphorylation sites confer reproducibility of the rod's single-photon responses

Although signals controlled by single molecules are expected to be inherently variable, rod photoreceptors generate reproducible responses to single absorbed photons. We show that this unexpected reproducibility-the consistency of amplitude and duration of rhodopsin activity-varies in a graded and systematic manner with the number but not the identity of phosphorylation sites on rhodopsin's C terminus. These results indicate that each phosphorylation site provides an independent step in rhodopsin deactivation and that collectively these steps tightly control rhodopsin's active lifetime. Other G protein cascades may exploit a similar mechanism to encode accurately the timing and number of receptor activation.     

A visual pigment with two physiologically active stable states

Red illumination of a Balanus amphitrite photoreceptor that has been adapted to blue light leads to prolonged depolarization in the late receptor potential. This depolarization can be switched off by further exposure to a blue stimulus. The early receptor potential in this cell is purely depolarizing or largely hyperpolarizing; the former is true if the cell has been adapted to red light, and the latter, if blue light has been used. The color-adaptation "memories" for both early and late receptor potentials appear to be permanent. The existence of two stable states for the early receptor potential directly implies a pigment with two stable states, and these apparently contribute antagonistically to the late receptor potential.     

Elementary response of olfactory receptor neurons to odorants

Signaling by heterotrimeric GTP-binding proteins (G proteins) drives numerous cellular processes. The number of G protein molecules activated by a single membrane receptor is a determinant of signal amplification, although in most cases this parameter remains unknown. In retinal rod photoreceptors, a long-lived photoisomerized rhodopsin molecule activates many G protein molecules (transducins), yielding substantial amplification and a large elementary (single-photon) response, before rhodopsin activity is terminated. Here we report that the elementary response in olfactory transduction is extremely small. A ligand-bound odorant receptor has a low probability of activating even one G protein molecule because the odorant dwell-time is very brief. Thus, signal amplification in olfactory transduction appears fundamentally different from that of phototransduction.     

Membrane origin of the fast photovoltage of squid retina

When a bright light flash is absorbed by a small region in the outer segments of squid photoreceptors fixed in glutaraldehyde, a brief pulse of membrane current flows locally. The passive spreading of this current along the outer segments produces the photochemical component of the "early receptor potential." The source of the current lies electrically in parallel with the cell membranes and perhaps is located within them. Fixation with glutaraldehyde apparently does not reduce the resistance of the cell membrane to less than 5 percent of its value in live cells.     

Movement of retinal along the visual transduction path

Movement of the ligand/receptor complex in rhodopsin (Rh) has been traced. Bleaching of diazoketo rhodopsin (DK-Rh) containing 11-cis-3-diazo-4-oxo-retinal yields batho-, lumi-, meta-I-, and meta-II-Rh intermediates corresponding to those of native Rh but at lower temperatures. Photoaffinity labeling of DK-Rh and these bleaching intermediates shows that the ionone ring cross-links to tryptophan-265 on helix F in DK-Rh and batho-Rh, and to alanine-169 on helix D in lumi-, meta-I-, and meta-II-Rh intermediates. It is likely that these movements involving a flip-over of the chromophoric ring trigger changes in cytoplasmic membrane loops resulting in heterotrimeric guanine nucleotide-binding protein (G protein) activation.     

Rescue of the Drosophila phototransduction mutation trp by germline transformation

Phototransduction is the process by which light-stimulated photoreceptor cells of the visual system send electrical signals to the nervous system. Many of the steps that follow the initial event in phototransduction, absorption of light by rhodopsin, are ill-defined. The fruitfly, Drosophila melanogaster, provides a means to dissect phototransduction genetically. Mutations such as transient receptor potential (trp) affect intermediate steps in phototransduction. In order to facilitate molecular studies of phototransduction, the trp gene was isolated and its identity was confirmed by complementing the mutant trpCM allele of the trp gene by P-element mediated germline transformation of a 7.1-kilobase DNA fragment. Expression of the trp gene begins late in pupal development and appears to be limited to the eyes and ocelli.     

Bacterial rhodopsin: evidence for a new type of phototrophy in the sea

Extremely halophilic archaea contain retinal-binding integral membrane proteins called bacteriorhodopsins that function as light-driven proton pumps. So far, bacteriorhodopsins capable of generating a chemiosmotic membrane potential in response to light have been demonstrated only in halophilic archaea. We describe here a type of rhodopsin derived from bacteria that was discovered through genomic analyses of naturally occuring marine bacterioplankton. The bacterial rhodopsin was encoded in the genome of an uncultivated gamma-proteobacterium and shared highest amino acid sequence similarity with archaeal rhodopsins. The protein was functionally expressed in Escherichia coli and bound retinal to form an active, light-driven proton pump. The new rhodopsin exhibited a photochemical reaction cycle with intermediates and kinetics characteristic of archaeal proton-pumping rhodopsins. Our results demonstrate that archaeal-like rhodopsins are broadly distributed among different taxa, including members of the domain Bacteria. Our data also indicate that a previously unsuspected mode of bacterially mediated light-driven energy generation may commonly occur in oceanic surface waters worldwide.     

All-Carbon Molecules: Evidence for the Generation of Cyclo[18]carbon from a Stable Organic Precursor

The unambiguous structural characterization of a single-sized all-carbon molecule requires its chemical synthesis. For cyclo[18]carbon, ab initio calculations predict a relatively stable, cyclic D9h ground state geometry with alternating C-C (1.36 angstroms) and C identical withC (1.20 angstroms) bonds. The synthesis and x-ray crystal structure of a direct precursor to C(18) are described. The analysis of laser flash heating experiments on this precursor by time-of-flight mass spectroscopy shows a sequence of retro-Diels-Alder reactions leading to C(18) as the predominant fragmentation pattern. Structural evidence is provided for the generation of an all-carbon molecule from a well-characterized organic precursor.     

Anabaena sensory rhodopsin: a photochromic color sensor at 2.0 A

Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms. Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported. We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions. Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein. The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer.     

beta-Arrestin: a protein that regulates beta-adrenergic receptor function

Homologous or agonist-specific desensitization of beta-adrenergic receptors is thought to be mediated by a specific kinase, the beta-adrenergic receptor kinase (beta ARK). However, recent data suggest that a cofactor is required for this kinase to inhibit receptor function. The complementary DNA for such a cofactor was cloned and found to encode a 418-amino acid protein homologous to the retinal protein arrestin. The protein, termed beta-arrestin, was expressed and partially purified. It inhibited the signaling function of beta ARK-phosphorylated beta-adrenergic receptors by more than 75 percent, but not that of rhodopsin. It is proposed that beta-arrestin in concert with beta ARK effects homologous desensitization of beta-adrenergic receptors.     

Electrical output of a receptor membrane

The electrical output of the receptor membrane of the nonmyelinated ending of Pacinian corpuscles is a function of the electrical gradients across the receptor membrane. The generator potential of the receptor membrane in response to equal mechanical stimuli varies linearly with the intensity of polarizing currents passed through the membrane. The production of a generator potential leaves a refractory state in the receptor membrane which is independent of the amount of charge transferred across the membrane but is dependent on a factor related to the strength of the stimulus which produced the response.