Low amplification and fast visual pigment phosphorylation as mechanisms characterizing cone photoresponses.

Vertebrate cone photoreceptors are known to show lower light sensitivity and briefer photoresponses than rod photoreceptors. To understand the molecular mechanisms characterizing cone photoresponses, we compared some of the reactions in the phototransduction cascade between rods and cones. For this purpose, rods and cones were obtained in quantities large enough to do biochemical studies. The cells were purified from the retina of carp (Cyprinus carpio) with a stepwise Percoll gradient. The purified rod fraction contained almost no other kinds of cells besides rods, and the purified cone fraction contained a mixture of red-, green-, and blue-sensitive cones in the ratio 3: approximately 1: approximately 1. We prepared membrane preparations from the rod and the cone fraction, and in these membranes, we measured activation efficiencies of the reactions in the phototransduction cascade. The results showed that the signal amplification is lower in the cone membranes, which accounts for the lower light sensitivity in cones. Furthermore, we measured the time courses of visual pigment phosphorylation. The result showed that the phosphorylation is much faster in the cone membranes, which also explains the lower light sensitivity and, in addition, the briefer photoresponse in cones.

Amino acid residues of S-modulin responsible for interaction with rhodopsin kinase.

S-modulin in frog or its bovine homologue, recoverin, is a 23-kDa EF-hand Ca(2+)-binding protein found in rod photoreceptors. The Ca(2+)-bound form of S-modulin binds to rhodopsin kinase (Rk) and inhibits its activity. Through this regulation, S-modulin is thought to modulate the light sensitivity of a rod. In the present study, we tried to identify the interaction site of the Ca(2+)-bound form of S-modulin to Rk. First, we mapped roughly the interaction regions by using partial peptides of S-modulin. The result suggested that a specific region near the amino terminus is the interaction site of S-modulin. We then identified the essential amino acid residues in this region by using S-modulin mutant proteins: four amino acid residues (Phe(22), Glu(26), Phe(55), and Thr(92)) were suggested to interact with Rk. These residues are located in a small closed pocket in the Ca(2+)-free, inactive form of S-modulin, but exposed to the surface of the molecule in the Ca(2+)-bound, active form of S-modulin. Two additional amino acid residues (Tyr(108) and Arg(150)) were found to be crucial for the Ca(2+)-dependent conformational changes of S-modulin.

Selective activation of G-protein subtypes by vertebrate and invertebrate rhodopsins.

We have quantitatively investigated specificities in activating G-protein subtype by bovine and squid rhodopsins to examine whether or not the phototransduction cascade in each of the photoreceptor cells is determined by the colocalization of a large amount of G-protein subtype (Gt or Gq). In contrast to the efficient activation of respective Gt and Gq, bovine and squid rhodopsins scarcely activated G-protein counterparts. Exchange of alpha- and betagamma-subunits of Gt and Gq indicated the critical role of the alpha-subunit in specific binding to respective rhodopsins. Thus the specific recognition of G-protein subtype by each rhodopsin is a major mechanism in determining the phototransduction cascade.

Identification of a new intermediate state that binds but not activates transducin in the bleaching process of bovine rhodopsin.

Using time-resolved low-temperature spectroscopy, we have examined whether or not bovine rhodopsin has a unique transducin-binding state, meta Ib, previously detected from chicken rhodopsin. Unlike chicken meta Ib, bovine meta Ib was detected only by detailed kinetics analysis of the bleaching process, but it was stabilized by transducin and visualized in the observed spectral changes. From the effect of GTPgammaS, it was revealed that meta Ib induced no GDP-GTP exchange reaction in transducin. Thus meta Ib is a common intermediate of vertebrate rhodopsin and transducin is activated in two steps by meta Ib and meta II.

Presence of two rhodopsin intermediates responsible for transducin activation.

To identify how many rhodopsin intermediates interact with retinal G-protein transducin, the photobleaching process of chicken rhodopsin has been investigated in the presence or absence of transducin by means of time-resolved low-temperature spectroscopy. Singular value decomposition (SVD) analysis of the spectral data showed that a new intermediate called meta Ib is present between formally identified metarhodopsin I (now referred to as meta Ia) and metarhodopsin II (meta II). Since the absorption maximum of meta Ib (460 nm) is similar to that of meta Ia (480 nm), but considerably different from that of meta II (380 nm), meta Ib should have a protonated retinylidene Schiff base as its chromophore. Whereas transducin showed no effect on the conversion process between lumirhodopsin (lumi) and meta Ia, it affected the process between meta Ia and meta Ib and that between meta Ib and meta II. These results suggest that at least two intermediates (meta Ib and meta II) interact with transducin. The addition of GTPgammaS had no effect on the meta Ib-transducin interaction, while it abolished the ability of transducin to interact with meta II. Thus, meta Ib only binds to transducin, while meta II catalyzes a GDP-GTP exchange in transducin. These results suggest that deprotonation of the Schiff base chromophore is not necessary for the binding to transducin, while changes in protein structure including Schiff base deprotonation are needed to induce the GDP-GTP exchange in transducin.

Photochemical and biochemical properties of chicken blue-sensitive cone visual pigment.

Through low-temperature spectroscopy and G-protein (transducin) activating experiments, we have investigated molecular properties of chicken blue, the cone visual pigment present in chicken blue-sensitive cones, and compared them with those of the other cone visual pigments, chicken green and chicken red (iodopsin), and rod visual pigment rhodopsin. Irradiation of chicken blue at -196 degrees C results in formation of a batho intermediate which then converts to BL, lumi, meta I, meta II, and meta III intermediates with the transition temperatures of -160, -110, -40, -20, and -10 degrees C. Batho intermediate exhibits an unique absorption spectrum having vibrational fine structure, suggesting that the chromophore of batho intermediate is in a C6-C7 conformation more restricted than those of chicken blue and its isopigment. As reflected by the difference in maxima of the original pigments, the absorption maxima of batho, BL, and lumi intermediates of chicken blue are located at wavelengths considerably shorter than those of the respective intermediates of chicken green, red and rhodopsin, but the maxima of meta I, meta II, and meta III are similar to those of the other visual pigments. These facts indicate that during the lumi-to-meta I transition, retinal chromophore changes its original position relative to the amino acid residues which regulate the maxima of original pigments through electrostatic interactions. Using time-resolved low-temperature spectroscopy, the decay rates of meta II and meta III intermediates of chicken blue are estimated to be similar to those of chicken red and green, but considerably faster than those of rhodopsin. Efficiency in activating transducin by the irradiated chicken blue is greatly diminished as the time before its addition to the reaction mixture containing transducin and GTP increases, while that by irradiated rhodopsin is not. The time profile is almost identical with those observed in chicken red and green. Thus, the faster decay of enzymatically active state is common in cone visual pigments, independent of their spectral sensitivity.

Single amino acid residue as a functional determinant of rod and cone visual pigments.

The visual transduction processes in rod and cone photoreceptor cells begin with photon absorption by the different types of visual pigments. Cone visual pigments exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (meta II) than does the rod visual pigment, rhodopsin, as expected, due to the functional difference between rod and cone photoreceptor cells. To identify the amino acid residue(s) responsible for the difference in molecular properties between rod and cone visual pigments, we selected three amino acid positions (64, 122, and 150), where cone visual pigments have amino acid residues electrically different from those of rhodopsin, and prepared mutants of rhodopsin and chicken green-sensitive cone visual pigment. The results showed that the replacement of Glu-122 of rhodopsin by the residue containing green- or red-sensitive cone pigment converted rhodopsin's rates of regeneration and meta II decay into those of the respective cone pigments, whereas the introduction of Glu-122 into green-sensitive cone visual pigment changed the rates of these processes into rates similar to those of rhodopsin. Furthermore, exchange of the residue at position 122 between rhodopsin and chicken green-sensitive cone pigment interchanges their efficiencies in activating retinal G protein transducin. Thus, the amino acid residue at position 122 is a functional determinant of rod and cone visual pigments.

Effect of chloride on the thermal reverse reaction of intermediates of iodopsin.

Among the intermediates in the bleaching process of iodopsin, a chicken red-sensitive cone visual pigment, the batho and meta I intermediates (batho and meta I) formed at low temperatures revert to the original iodopsin by thermal reactions [Yoshizawa & Wald (1967) Nature 214, 566-571; Imamoto, Imai, Yoshizawa, & Shichida (1994) FEBS Lett. 354, 165-168]. In order to elucidate the relationship between Cl- binding to iodopsin and these reverse reactions, we have prepared a sample of iodopsin whose Cl(-)-binding site is vacant (anion-unbound iodopsin) and compared the thermal reactions of its batho and meta I intermediates with those of Cl(-)-bound (native) and nitrate-bound iodopsins. The reverse reaction from batho is observed in both Cl(-)-bound and anion-unbound iodopsins, while the reaction from meta I is observed only in Cl(-)-bound iodopsin. These results indicate that Cl- binding is indispensable for the reverse reaction from meta I, but not from batho. The reverse reaction from meta I has been further investigated as a function of Cl- concentration, and the dissociation constant of Cl- in meta I is estimated to be approximately 20 mM. This value is about 200 times larger than that of iodopsin (0.1 mM), and close to the physiological Cl- concentration in photoreceptor cells, suggesting that Cl- could be released from the protein moiety during the bleaching of iodopsin.