Neuron,

Vol. 8, 465-472, March, 1992, Copyright

0 1992 by Cell Press

Histidine Residues Regulate the Transition of Photoexcited Rhodopsin to Its Active Conformation, Metarhodopsin II Charles J. Weitz* and Jeremy Nathans*+ *Department of Molecular Biology and Genetics and Department of Neuroscience +The Howard Hughes Medical Institute Johns Hopkins University School of Medicine Baltimore, Maryland 21205

Summary The biologically active photoproduct of rhodopsin, metarhodopsin II (M II), exists in a pH-sensitive equilibrium with its precursor, metarhodopsin I (M I). Increasing acidity favors M II, with the midpoint of the pH titration curve at pH 6.4. To test the long-standing proposal that histidine protonation regulates this conformational transition, we characterized mutant rhodopsins in which each of the 6 histidines was replaced by phenylalanine or cysteine. Only mutants substituted at the 3 conserved histidines showed abnormal M I-M II equilibria. Those in which His-21 1 was replaced by phenylalanine or cysteine formed little or no M II at either extreme of pH, whereas mutants substituted at His-65 or at His-152 showed enhanced sensitivity to protons. The simplest interpretation of these results is that His-21 1 is the site where protonation strongly stabilizes the M II conformation and that His-65 and His-152 are sites where protonation modestly destabilizes the M II conformation. Introduction Vision in dim light is mediated by rhodopsin, the visual pigment of retinal rod photoreceptors (Wald, 1968). The sole action of light in visual excitation is the photoisomerization of the Al-cis-retinal chromophore of rhodopsin to the all trans configuration; all subsequent processes are thermal in nature (Hubbard and Kropf, 1958). The highly energetic primary photoproduct, formed within 200 fs (Schoenlein et al., 1991), relaxes via a series of spectrally distinguishable intermediates to metarhodopsin II (M II) (h,,, = 380 nm), the last intermediate to form rapidly enough to play a role in the initiation of the rod photoreceptor response (Wald, 1968). Evidenceaccumulated in the last decade indicates that M II is the active form of photoexcited rhodopsin. M II binds specifically to the G protein transducin (Bennett et al., 1982; Emeis et al., 1982) and catalyzes its conversion to the GTP binding form (Kibelbeketal., 1991),therebyactivatingthe biochemical cascade of vision (Stryer, 1986). M II exists in an equilibrium with its immediatespectral precursor metarhodopsin I (M I) (h,,,= 478 nm), an equilibrium that is stable for tens of minutes at temperatures near O°C (Mathews et al., 1963). This equilibrium is dependent on temperature, with increasing temperature favoring M II, and on pH, with a nearly complete shift toward M II at pH 5 and a

nearly complete shift toward M I at pH 8. The midpoint of the titration curve is at pH 6.4, prompting nearly 30 years ago the proposal that protonation of an imidazole side chain of a histidine residue might be tightly coupled to the M I to M II transition (Mathews et al., 1963). The slope of the pH titration curve has been repeatedly observed to be shallower than the theoretical slope for a single protonation site (Mathews et al., 1963; Parkes and Liebman, 1984), raising the possibility that concurrent protonation at oneor more additional sites, possibly also on histidine residues, weakly favors the M I conformation. Direct measurements of pH changes following rhodopsin photoactivation are consistent with the pH dependenceof theM I-M II equilibrium. Light-dependent proton uptake by rhodopsin, measured by pH electrode, had a pKA of 6.6, also suggestive of histidine protonation (Radding and Wald, 1956). An optical study using a pH-sensitive dye determined that a net of one proton is taken up from solution by rhodopsin duringthecourseoftheM I toM II transition (Bennett, 1980). This result suggests that at least two protons are taken up into opsin (the apoprotein of rhodopsin), since the protonated Schiff’s base linkage of the chromophore to Lys-296 of opsin loses its proton during the M I-M II transition (Doukas et al., 1978). To test the hypothesis that protonation of one or more histidine residues is coupled to the M I to M II transition and the subsidiary hypothesis that protonation of one or more additional histidine residues negatively modulates this transition, we used site-directed mutagenesis and cDNAexpression in 293s cells to prepare a set of seven mutant bovine rhodopsins for study by spectroscopy. Bovine rhodopsin contains 6 histidine residues (Figure 1) (Ovchinnikov et al., 1983; Hargrave et al., 1983; Nathans and Hogness, 1983), of which 3 (His-65, His-152, His-211) are conserved in all vertebrate rhodopsins known to date (human: Nathans and Hogness, 1984; sheep: Findlay and Pappin,1986;mouse:Baehretal.,1988;chicken:Takao et al., 1988). In each mutant rhodopsin we replaced 1 histidine residue by phenylalanine, a residue similar to histidine in bulk and hydrophobicity but which cannot carry a positive charge in the relevant pH range. In addition, we replaced His-211 by cysteine, the residue found in the homologous position in all vertebrate cone visual pigments known to date (human: Nathans et al., 1986; nonhuman primates: Neitz et al., 1991; chicken: Kuwata et al., 1990; fish: Yokoyama and Yokoyama, 1990). Results All of the Expressed Mutant Opsins Form Visual Pigments That Have Normal Properties in the Ground State Since improperly folded or grossly perturbed opsins

NelJVJn 466

base linkage temperature

Figure 1. Model of the Topology of Bovine Rhodopsin in the ROS Disc Membrane (the C-Terminus Lies on the Cytoplasmic Side) Numbered amino acids indicate the positions of the 6 histidine residues, and asterisks mark the 3 histidine residues conserved in vertebrate rhodopsins known to date. Also shown are Lys-296, the amino acid whose side chain forms the retinylidene Schiff’s base linkage, and Clu-113, the amino acid proposed to stabilize the protonated form of the Schiff’s base (see text).

would not be expected to form a binding pocket for the chromophore, we tested the spectral properties of each mutant rhodopsin after reconstitution in vitro with II-cis-retinal to verify that each amino acid substitution was tolerated by the protein. The photobleaching absorption difference spectra in Figure 2 indicate that each expressed mutant opsin reconstitutes with II-cis-retinal to form a photolabile visual pigment and that the h,,, of each mutant does not differ significantly from that of expressed wild-type rhodopsin (WT). These results indicate that each amino acid substitution has little or no effect on the interaction between the opsin and the chromophore in the ground state or on the release of all-trans-retinal consequent to photobleaching at 25% The release of the chromophore suggests that each mutant rhodopsin can form at least some M II at 25%, since studies of a rhodopsin derivative blocked in an M l-like conformation suggest that in M I the retinylidene Schiff’s

WAVELENGTH Figure 2. Photobleaching

Absorption

Difference

Spectra

is resistant to hydrolysis (Longstaff et al., 1986).

even

at room

M I-M II Equilibria of Mutant Rhodopsins Substituted at Conserved Histidines Show Abnormal Responses to Changes in pH Following irradiation of rhodopsin near O°C, the hydrolysis of M II is so slow that the M I-M II equilibrium is stable for tens of minutes, permitting conventional spectroscopic study (Mathews et al., 1963). Absorption spectra showing the pH sensitivity of the M i-M II equilibrium (2°C) of rhodopsin extracted from bovine rod outer segment (ROS) membranes are illustrated in Figure 3 (upper left). Prior to irradiation (-hv), the spectrum reflects the ground state absorption of rhodopsin (h,,, = 498 nm); after irradiation and attainment of equilibrium (within a few milliseconds; Mathews et al., 1963), aliquots adjusted to different pH show the expected distribution of M I and M II, with the most alkaline sample (pH 7.9) showing a nearly complete shift to M I and the most acidic sample (pH 5.6) showing a nearly complete shift to M II. Because the avaiiable quantity and optical purity of expressed rhodopsins were much less than that of rhodopsin from ROS, we used an absorption difference technique to monitor the M I-M II equilibrium of expressed rhodopsins because of its superior signal to noise ratio and insensitivity to photostable contaminants. Figure 3 (lower left) shows the family of absorption difference spectra derived from the control data shown in Figure 3 (upper left); photoreactants are represented as positive peaks and photoproducts as negative peaks. Since at pH 7.9 the predominant photoproduct is M I, the absorption spectrum of which largely overlaps that of rhodopsin, the difference absorption spectrum shows a small positive peak &,,,, approximately 525 nm) corresponding to that portion of the rhodopsin spectrum that minimally overlaps the M I spectrum and a shallow negative peak (h,,, approximately 460 nm) corresponding to the part of

(nm) of Expressed

Rhodopsins

Solubilized

in Digitonin

All were prepared from cell lines stably expressing the indicated opsin, except for H195F, which was prepared from transientlytransfected cells. The spectra are representative of at least three independent experiments for each expressed rhodopsin. Similar photobleaching difference spectra for HZIIF and H211C have been previously reported (Nathans, 1990a).

Histidines

Regulate Rhodopsin

Activation

467

0.187

WT PH=5.8,

O.C?JO

\

pn=7.5

m:

0.0 2 to+ t5 Li

p,r”,~

a

0.0 yj%

lljzgfy~~~;~

-0.8, 300

500

700

300

500

MO

WAVELENGTH Figure 3. Absorption Spectra Solubilized in Digitonin

Demonstrating

300

500

700

Xx)

500

700

(nm)

the Effect of pH on the M I-M

II Equilibrium

of Wild-Type

and Mutant

Rhodopsins

(Upper Left) The absorption spectrum of rhodopsin from ROS prior to irradiation (-hv) and a set of absorption spectra from parallel samples after irradiation and attainment of the M I-M II equilibrium (+hv, pH as indicated). The spectra have not been corrected for the absorption due to photoregenerated rhodopsin and isorhodopsin (Mathews et al., 1963). (Lower Left) The family of M I-M II equilibrium difference spectra derived from the data in the upper left panel. Each is derived by subtracting the postirradiation absorption spectrum from the preirradiation absorption spectrum (see text). (Remaining Panels)The M I-M II equilibrium difference spectra obtained at pH 5.8 and at pH 7.8 for the expressed rhodopsins prepared from stably expressing cell lines (all except H195F) or transiently transfected cells (H195F). To facilitate direct comparison, curves have been scaled such that the photobleaching difference peak for the total visual pigment (data not shown, but nearly the same in appearance as that of the M I-M II difference curve for wild-type rhodopsin at pH 5.8) would occupy the same proportion of area in each panel. The increased noise and baseline drift seen at the UV end of many of the spectra are due, respectively, to the large absorbances of the samples in the UV and to changes in light scattering that occur during the course of the measurements, which disproportionately affect the shorter wavelengths. Each panel is representative of at least three independent experiments. In addition, the same results were obtained for H211F with three independent cell lines; the same results were obtained for H211C with two independent cell lines and with transiently transfected cells.

the M I spectrum that minimally overlaps the rhodopsin spectrum. There is no detectable negative peak corresponding to the M I I photoproduct (I.,,, approximately 385 nm under these conditions). In the pH 5.6 absorption difference spectrum, the positive rhodopsin peak is largely undistorted because there is little M I present, and a large negative peak appears, corresponding to M II. Despite their similarity, difference spectra showing the presence of M II can be distinguished from those showing the presence of released all-trans-retinal (such as after irradiation at 25’C or after denaturation of rhodopsin) by virtue of the fact that under these conditions all-trans-retinal has a h,,, that is approximately5-8 nm less than that of M II and has a lower molar absorptivity (all-trans-retinal: 39,000; M II: 42,000; Mathews et al., 1963). By these criteria, all of the negative peaks in the UV in Figure 3, Figure 4, and Figure 5 very likely represent M II. Absorption difference spectraof M I-M II equilibria obtained from expressed rhodopsins at pH 5.8 and pH 7.8 are shown in Figure 3. Expressed wild-type rhodopsin shows the expected nearly complete shift

toward M II at pH 5.8, but unlike rhodopsin from ROS (compare with Figure 3, lower left, pH 7.9), it shows a small amount of M II at alkaline pH, more than can be accounted for by the slight buffering of pH seen in the samples of expressed rhodopsins (see Experimental Procedures). Since rhodopsin from ROS shows this same behavior when diluted in membranes from 293s cells and solubilized in digitonin (data not shown), the difference is probably due to the high concentration of 293s membranes or to the unusual composition of ROS membranes (Daemen, 1973). The negative control spectra, from an otherwise identical cell line stably transfected with an expression plasmid lacking a rhodopsin cDNA insert, show the background at the same scale. The mutant rhodopsins substituted at the 3 histidine residues that are not conserved in known vertebrate rhodopsins (HIOOF, H195F, H278F) show spectra that are indistinguishable from that of wildtype rhodopsin, indicating that those histidine residues do not appreciably influence the M I-M II equilibrium. In contrast, all of the mutant rhodopsins substituted

Neuron 468

at the 3 conserved histidine residues (H65F, H152F, H211F, H211C) show M I-M II equilibrium spectra different from that of wild-type rhodopsin. Both H65F and H’l52Fgenerate M I-M II equilibria that are sensitive to pH, but H65F forms considerably more M II, and H152F forms slightly more M II than does wildtype rhodopsin at pH 7.8 (Figure 3). Although the pH 7.8differencecurveforH152Fappearsonlymarginally different from that of wild type, a comparison of spectra from three independent experiments shows that the H152F equilibrium is significantly shifted toward M II at pH 7.8 (wild-type h,,, = 513 + 1.6 nm [SD]; H152F h,,, = 507 & 1.6 nm; P = 0.01; compare with peak maxima in difference spectra from ROS rhodopsin controls, Figure 3, lower left). These results indicatethat in wild-type rhodopsin theM II conformation is moderately destabilized by His-65 and slightly destabilized by His-152. Both H211F and H211C show no detectable M II and no detectable sensitivity to pH, with difference spectra indicating the presence of only M I at both pH values (Figure 3). To test this conclusion by an independent method, we measured the residual rhodopsin plus isorhodopsin present immediately after M I-M II equilibria were established (Mathews et al., 1963). Because the samples were irradiated with 580 to nm light, M I (h,,, = 478 nm) is photoregenerated rhodopsin plus isorhodopsin much more efficiently and Kropf, 1958); than M II (h,,, = 380 nm) (Hubbard therefore, at photo steady state the fraction of original rhodopsin present as photoregenerated rhodopsin plus isorhodopsin is proportional to the amount of M I at equilibrium (Baldwin and Hubbell, 1985). Table 1 shows the measured values at pH 5.8 and pH 7.8 for expressed wild-type rhodopsin and H211F. As expected, for wild-type rhodopsin there was a significantly greater photoregeneration of rhodopsin plus isorhodopsin in the samples at pH 7.8. For H211F, the values at pH 5.8 and 7.8 show no significant difference, and these values are considerably higher than those of wild-type rhodopsin. Thus, analysis of photoregeneration efficiencies also indicates that irrespective of pH H21’lFgeneratedessentiallyonlyM I.Theseresults indicate that in wild-type rhodopsin His-211 makes a large contribution to the stabilization of M Il.

geststhat His-211 is not obligatoryfortheformation of M II. To test this suggestion directly, we used reagents that are known to shift the equilibrium toward M II even at alkaline pH. Figure 4 shows the effect on the M I-M II equilibrium produced by addition of the detergent B-octyl glucoside (BOG) to the digitoninmembrane micelles in which wild-type rhodopsin or H211F had been solubilized. Previous studies have shown that B O G shifts the equilibrium toward M II, largely by producing a 20-fold increase in the rate of the forward reaction (Konig et al., 1989). For ROS rhodopsin (pH 7.9) and expressed wild-type rhodopsin (pH 7.8), B O G produced a concentration-dependent shift of the equilibrium toward M II, with 2% B O G sufficient to produce a nearly complete shift. At 2% B O G H211F showed a moderate but unambiguous shift toward M II, forming approximately as much M II as did wild-type rhodopsin in the absence of BOG. This experiment indicates that His-211 is not obligatory for M II formation. The direct observation of M II formed by H211F also argues against the unlikely possibility that replacing His-211 merely prevents the absorption changes that accompany the M I to M II transition without affecting the actual conformational transition. The M I-M II Equilibrium of Mutant Rhodopsin H211 F Shows No Detectable Shift Toward M II in Response to a Peptide Derived from Transducin Figure 5 shows the effect produced by a synthetic peptide (IKENLKDCGLF) corresponding to the C-ter-

A Histidine at Position 211 Is Not Required for M II Formation The release of all-trans-retinal by H211F and H211C consequent to photobleaching at 25’C (Figure 2) sug-

Table 1. Photoregenerated

Wild type H211F

Rhodopsin

and lsorhodopsin

pH 5.8

pH 7.8

P Value

12 f 3.1 (SD) 32 + 4.7

21 f 2.2 36 k 3.6

0.016 0.32

Photoregenerated rhodopsin plus isorhodopsin as a percentage of total visual pigment in the sample. Each value is derived from three independent experiments.

Figure 4. Absorption fect of the Detergent M I-M II Equilibrium

Difference Spectra Demonstrating DOG (Percent [w/v] as Indicated)

the Efon the

Rhodopsin from ROS (upper), expressed wild-type rhodopsin (middle), and expressed mutant rhodopsin H211F (lower). Prior to addition of BOG all samples had been soiubilized in digitonin (final concentration of 2%).

Histidines

Regulate Rhodopsin

Activation

469

the reagent that shifts the equilibrium by increasing the forward rate. The failure of H211F and H211C to respond to the peptide with a similar shift toward M II raises the possibilitythat in the mutant pigments the forward rate is so slow that equilibrium was not established. Discussion

-o.066m~j 700

WAVELENGTH Figure 5. Absorption Difference Spectra Demonstrating fect of Synthetic Peptide IKENLKDCGLF (Concentration cated) on the M I-M II Equilibrium

the Efas Indi-

Rhodopsin from ROS (upper), expressed wild-type rhodopsin (middle), and expressed mutant rhodopsin H211F (lower). Prior to addition of peptide, all samples were solubilized in digitonin (final concentration of 2%). This peptide has been previously shown to stabilize the M II form of rhodopsin from ROS (Hamm et al., 1989); its sequence corresponds to the 11 C-terminal residues of bovine rod transducin.

minal 11 residues of the a subunit of bovine rod transducin, a region shown by competition binding studies to form part of the binding site for M II on transducin (Hamm et al., 1989)This peptidecan mimic transducin in specific binding to M II, shifting the equilibrium toward M II without significantly affecting the rate of the forward reaction (Hamm et al., 1989), presumably by forming stable complexes with M II and thereby decreasing the rate of the reverse reaction. For ROS rhodopsin (pH 7.9) and expressed wild-type rhodopsin (pH 7.8), this peptide produced a concentrationdependent shift of the M I-M II equilibrium toward M II, with 3 m M peptide sufficient to produce a nearly complete shift in both cases. H211F (pH 7.8) shows virtually no detectable M II at 3 m M peptide, suggesting that any M II formed by H211F in the absence of peptide must be far below the detection limit. H211C showed the same lack of response to 3 m M peptide (data not shown). These results suggest that H211F and H211C would exhibit little or no biological activity. Of twoconditions that show similar potency in shifting the equilibrium of wild-type rhodopsin toward M II (3 m M peptide and 2% DOG), it is of interest that H211F forms detectable M II only in response to DOG,

The results described above indicate that in wild-type rhodopsin His-211 makes a large contribution to the stabilization of M II and that His-65 and His-152 in part oppose this stabilization. Given the dependence on pH suggesting that histidine protonation might be coupled to the M I to M II transition (Mathews et al., 1963), the most likely interpretation of our results is that His-211 is the site of protonation strongly stabilizing M II and that His-65 and His-152 are additional sites of protonation weakly destabilizing M II, as suggested by the shallow slope of the titration curve (Mathews et al., 1963; Parkes and Liebman, 1984). Although His211 is predicted to lie within a transmembrane segment(Figure I), it need not beconsidered inaccessible to protons from the aqueous solvent-in M II the retinylidene Schiff’s base linkage to Lys-296 becomes susceptible to attack by hydroxylamine (Falk and Fatt, 1968), indicating that the binding pocket for the chromophore is accessible from the surrounding solution. Our experiments do not exclude the possibility that residues other than histidines 65, 152, and 211 also bind protons, nor do they exclude the possibility that one or more of these histidine residues is not itself a site of protonation, but instead acts to modify the pKA of one or more nonhistidine residues. If, as seems likely, His-211 is the critical site of protonation, by what specific mechanisms might its protonation favor the formation of M I I or stabilize it? The M I to M II transition is known to be accompanied by deprotonation of the Schiff’s base linkage of the retinal chromophore at Lys-296 (Doukas et al., 1978), an event apparently obligatory for both the UV absorption maximum of M II and its biological activity (Longstaff et al., 1986). Recent mutagenesis experiments strongly implicate Glu-113 as the counterion that stabilizes protonation of the retinylidene Schiff’s base in the ground state of rhodopsin (Sakmar et al., 1989; Zhukovsky and Oprian, 1989; Nathans, 1990b). Remarkably, the E113Q mutant rhodopsin can bind all-trans-retinal and activate transducin in the absence of light (Sakmar et al., 1989), suggesting that an unprotonated retinylidene Schiff’s base (consequent to the absence of the Glu-113 carboxylate) is of itself sufficient to drive the protein to an M II-like conformational state if all-trans-retinal occupies the binding pocket. It seems reasonable to propose, therefore, that protonation of His-211 favors the M II conformation by destabilizing the ionic interaction between Glu-113 and the protonated retinylidene Schiff’s base linkage at Lys-296. This destabilization could be achieved by

Neuron 470

MII

MI

:N

L/-c NH

-

w

glu”” lyS2g6

NH

MII

NH %A

HN+ ;v

glu”3 IyS2g6

MI

:N

of M II by l-lis-65 and His-152 is in fact due to the protonation of those residues, then up to four popuiations of M I could exist, each defined by a unique state of protonation or unprotonation at these histidines and each perhaps undergoing the transition to M II at a different rate. This hypothesis predicts that a mutant rhodopsin substituted at both His-65 and His-152 will exhibit simple first order kinetics of M l decay and M II formation under conditions in which wild-type rhodopsin exhibits multiple first order kinetics.

-

HN+ %A

NH

X+ X+ Figure 6. Examples of Two General Mechanisms by Which tonation of His-211 Might Favor the M II Conformation

Pro-

(A) Direct electrostatic mechanism. Protonated His-211 electrostatically disrupts ion pairing between Glu-113 and the protonated retinylidene Schiff’s base linkage at Lys-296, leading to deprotonation of the Schiff’s base. (B) Indirect mechanism. Protonated His-211 interacts electrostatically with a charged or polar target residue (symbolized by X) elsewhereintheprotein,providingthedrivingforceforaconformational rearrangement that consequently disrupts ion pairing between Glu-113 and the protonated retinylidene Schiff’s base. Residue X could be any charged or polar amino acid; it is shown as positively charged for illustrative purposes only.

at least two general mechanisms. In the first (Figure 6A), protonated His-211 directly disrupts ion pairing between Glu-113 and the protonated Schiff’s base at Lys-296 by electrostatic interference. In the second (Figure 6B), protonated His-211 interacts electrostatically with a charged or polar amino acid “target” elsewhere in the protein, which provides the driving force for a conformational rearrangement that consequently destabilizes ion pairing between Glu-113 and the protonated Schiff’s base at Lys-296, perhaps by increasing the distance between them or altering their orientations. This model predicts that a mutant rhodopsin in which the charged or polar “target” residue has been replaced by a neutral or nonpolar one will behave similarly to H211F and H211C. In a variation on this indirect mechanism, the driving force for the conformational rearrangement might come from the unfavorable energetics of maintaining protonated His-211 in a hydrophobic environment as compared with a conformation in which its charged imidazole side chain is stabilized in a more polar environment. The possibility that M I and M II may each exist in multiple forms has been suggested by the complex kinetics of M I decay and M II formation (Wulff et al., 1958; Stewart et al., 1975; Hoffman et al., 1978; Straume et al., 1990), although the results of some studies are consistent with simple kinetics (King and Cutfreund, 1984) or suggest that the apparent multiplicity of processes depends on the experimental conditions (Applebury et al., 1974). If the modest destabilization

Electrical responses from rods show as much as IOOfold greater sensitivity to light than those from cones (Baylor, 1987). The remarkable photosensitivity of rods, in some cases sufficient for the detection of responses following the photoactivation of a single visual pigment molecule by one quantum of light (Baylor et al., 1979), puts unique constraints on the thermodynamic properties of rhodopsin (Baylor et al., 1980). Recent evidence strongly suggests that the thermodynamic properties of rhodopsin set the absolute behavioral threshold for the detection of dim flashes (Aho et al., 1988). Might control of the transition to the active form by His-211 represent a specialized adaptation of rhodopsin? It is intriguing that all known vertebrate cone visual pigments have a cysteine residue at the position homologous to His-211 in rhodopsin.Todate intermediates of cone pigment photoactivation analogous to thoseof rhodopsin have been spectroscopicallydocumented only up to M I (Hubbard and Kropf, 1959), although preliminary experiments have suggested that chicken iodopsin may form an M II-like intermediate (Hubbard et al., 1965). If the biologically active conformation of cone pigments is indeed analogous to M II, the presence of a cysteine residue in place of His-211 of rhodopsin raises the possibility that cone pigments might form M II by a mechanism different from thatof rhodopsinorthattheymightform it inefficiently, like the mutant rhodopsin H211C. If cone pig., ment M I is labileor subject to inactivation, then inefficient formation of cone pigment M I I could contribute to the difference between cones and rods in sensitivity to light. Experimental

Procedures

In Vitro Mutagenesis Covalently closed double-stranded pCIS-Rho expression plasmids encoding mutant bovine opsins were prepared as described (Nathans, 1990a) using oligonucleotide-directed mutagenesis followed by transformation of E. coii strain Mut L with the mutagenesis products, excision of the opsin coding region and its ligation into the parental pClS piasmid, transformation of wild-type E. coli, and colony blot hybridization to identify bacterial transformants harboring the mutant plasmids. The entire opsin coding region of each plasmid was sequenced on one strand by the dideoxy method to verify the expected mutation and to exclude any unintended ones. Transient Transfection and Isolation of Cell Lines Stably Expressing Mutant Bovine Opsins For transient transfections, 20-80 plates (IO cm) of 293s cells (a human embryonic kidney cell line) were cotransfected with mutant pCIS-Rho and pRSV-Tag by the calcium phosphate method and harvested 4872 hr later by incubation with 5 m M EDTA in

Histidines 471

Regulate Rhodopsin

Activation

phosphate-buffered saline, all as described (Nathans, IYYOa). Cell lines that stably express mutant opsins were prepared as described (Nathans et al., 1989) by cotransfecting 293s cells with mutant pCIS-Rho and pSV2neo, subjecting the transfectants to selection with C-418, and cloning resistant colonies. For selection of high expressing cell lines comparable to cRho 6, the cell line that expresses wild-type bovine opsin at 2-3 x 1Oh opsins per cell (Nathans et al., 1989), IO-50 C-418-resistant cell lines from each transfection were screened for opsin immunoreactivity (monoclonal antibody B6-30) byfluorescenceactivated cell sorting and/or by immunoblotting (Nathans et al., 1989); cell lines that were positive for opsin immunoreactivity were then screened by photobleaching difference absorption spectroscopy after reconstitution of the visual pigments by incubation of solubilized cell membranes with II-cis-retinal (see below). For cell lines expressing any given mutant opsin, only those that originated from G-418.resistant colonies on different plates are described as independent. Preparation of Rhodopsin from Cells Expressing Opsin Purified membranes from transientlytransfected cells harvested from 10 cm plates or from stable transfectants harvested from suspension culture in I-L spinner flasks (Nathans et al., 1989) were prepared as described (Nathans, 1990b) by cell homogenization and sucrose density ultracentrifugation, except that the nuclear pellet was resuspended in the low sucrose buffer for an additional sucrose density ultracentrifugation step. The membranes collected at the sucrose density interface were diluted 6-to 8-fold and pelleted by ultracentrifugation as described (Nathans, 199Ob). Membrane pellets were solubilized in 0.4-1.5 ml (1.5 ml for membranes from 1 liter of saturated suspension culture) of 2.396-2.996 digitonin (Kodak) in water, depending on the anticipated dilution of the sample by reagents yet to be added. All subsequent procedures were performed in the dark or under dim red light. Dry II-cis-retinal (stored under argon at -80°C) was dissolved in ethanol (to -5 mM) and added to the solubilized membranes (I-3 ul per ml) such that the final concentration was 3-to 5-fold greater than that estimated for the opsin. The samples were rotated end over end in the dark at 23OC for 4-6 hr, and insoluble material was removed by centrifugation in an Eppendorf microfuge at 14,000 rpm for 10 min at 4OC. ROS membranes were prepared as described (Papermaster and Dreyer, 1974). Membranes containing 0.5-1.0 mg of rhodop sin weresolubilized in 1-2 ml of 2.3%-2.9% digitonin, and insoluble material was removed as above. UV-Visible Absorption Spectroscopy For photobleaching absorption difference spectra (Figure 2), sodium phosphate buffer was added to a 308 PI aliquot of solubilized visual pigment (final concentration: 0.15 M sodium phosphate [pH 6.41, 2% digitonin); the sample was placed in a water-jacketed cuvette holder (25°C); the temperature of the sample was measured after 2 min (all samples were within 24.5OC-25.5OC); and four successive spectra were recorded (Kontron Instruments Uvikon 860). The sample was irradiated for 5 min with a 150 W lamp equipped with a fiber-optic guide (Chiu Technical) placed -0.5 cm above a 580 nm narrow band pass filter mounted on top of the cuvette holder. Four successive spectra were again recorded, and the difference spectrum was calculated by subtracting the averaged postirradiation spectrum from the averaged preirradiation spectrum. No significant changes in difference spectra were found upon additional irradiation. For M I-M II equilibrium absorption difference spectra (Figure 3), aliquots from a sample of solubilized visual pigment were distributed into tubes containing sodium phosphate buffer of pH 5.6, 6.4, and 7.9 (final concentration: 0.15 M sodium phosphate, 2% digitonin). Unlike the much purer ROS rhodopsin samples, the measured pH values of the solubilized expressed rhodopsin samples were slightly shifted (pH 5.8 f 0.1,6.5 f 0.1, 7.8 * O.l), presumably because of buffering by the much higher concentration of membranes and cellular proteins. The pH 6.5 aliquot in each case was used for a photobleaching control at

25°C as above. M I-M II equilibrium difference spectra were obtained as were the photobleaching spectra above, except that the circulating water bath was set at l°C-2’=‘C. Samples were equilibrated to temperature for 4 min, at which point the measured temperature of all samples was within 1.6°C-2.40C. The samples were irradiated through a 580 nm narrow band pass filter as described above; sample temperatures did not change by more than 0.2”C during the 5 min irradiation. Experiments in which BOG and peptide were used (Figure 4 and Figure 5, respectively) differed only in that all aliquots of solubilized visual pigment (200-300 ul) were added to sodium phosphate buffer of pH 7.9 plus either water, J3OC. or peptide (final concentration: 2% digitonin, 0.15 M sodium phosphate, and peptide and DOG as indicated). Measurement of photoregenerated rhodopsin plus isorhodopsin was performed as described (Mathews et al,, 1963). For accurate estimation of peak maxima, the best-fitting fifth order polynomial was calculated for selected wavelength ranges from differencespectrausingaMacintosh IICXequippedwithCricket Graph software. Acknowledgments We wish to thank C. Gorman, A. Levinson, and R. Kline for the pClS vector and 293s cell line, L. Stryer and Hoffmann-La Roche Inc. for II-cis-retinal, P. Hargrave for MAb B&30, K. P. Hofmann, J. Korenbrot, Y. Koutalis, and G. Yellen for helpful discussions and I. Chiu, S. Merbs, K.-Y. Yau, and C. Yellen for critical comments on the manuscript. C. J. W. is a recipient of a Physician Scientist Award from the National Eye Institute. This work was supportedbytheHowardHughesMedicallnstituteandtheNlH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

October

1, 1991; revised

December

11, 1991.

References Aho, A.-C., Donner, K., Hyden, C., Larsen, L. O., and Reuter, T. (1988). Low retinal noise in animals with low body temperature allows high visual sensitivity. Nature 334, 348-350. Applebury, M. L., Zuckerman, D. M., Lamola, A. A., and Jovin, T. M. (1974). Rhodopsin. Purification and recombination with phospholipids assayed by the metarhodopsin I-metarhodopsin II transition. Biochemistry 73, 3448-3458. Baehr, W., Falk, J. D., Bugra, K., Triantafyllos, J.T., and McGinnis, J. F. (1988). Isolation and analysis of the mouse opsin gene. FEBS Lett. 238, 253-256. Baldwin, P. A., and Hubbell, W. L. (1985). Effects of lipid environment on the light-induced conformational changes of rhodop sin. 1. Absence of metarhodopsin II production in dimyristoylphosphatidylcholinerecombinant membranes. Biochemistry24, 2624-2632. Baylor, D. A. (1987). Photoreceptor Ophthalmol. Vis. Sci. 28, 34-47.

signals

and vision.

Invest.

Baylor, D. A., Lamb, T. D., and Yau, K.-W. (1979). The membrane current of single rod outer segments. J. Physiol. 288, 589-611. Baylor, D. A., Matthews, G., and Yau, K.-W. (1980). Two components of electrical dark noise in toad retinal rod outer segments. J. Physiol. 309, 591-621. Bennett, N. (1980). Optical studyofthe light-induced protonation changes associated with the metarhodopsin II intermediate in rod-outer-segment membranes. Eur. J. Biochem. 777,99-103. Bennett, N., Michel-Villaz, M., and Kuhn, H. (1982). Light-induced interaction between rhodopsin and the CTP-binding protein. Eur. J. Biochem. 727, 96-103. Daemen, Biochim.

F. (1973). Vertebrate rod outer Biophys. Acta 300, 255-288.

segment

membranes.

NelJKJn 472

Doukas, A. G., Aton, B., Callender, R. H., and Ebrey, T. C. (1978). Resonance raman studies of bovine metarhodopsin I and metarhodopsin II. Biochemistry 17, 2432-2435. Emeis, D., Kuhn, H., Reichert, J., and Hofmann, K. P. (1982). Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium. FEBS Lett. 743, 29-34. Falk, G., and Fatt, P. (1968). Conductance light in rod outer segments. J. Physiol.

changes produced 798, 647-699.

by

Findlay, J. 6. C., and Pappin, D. J. C. (1986). The opsin family proteins. Biochem. J. 238, 625-642.

of

Hamm, H. E., Deretic, D., Arendt, A., Hargrave, P. A., Koenig, B., and Hofmann, K. P. (1989). Site of C protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit. Science 241, 832-835. Hargrave, P. A., McDowell, J. H., Curtis, D. R., Wang, J. K., Juszczak, E., Fong, S.-L., Mohanna-Rao, J. K., and Argos, P. (1983). The structure of bovine rhodopsin. Biophys. Struct. Mech. 9, 235244. Hoffman, W., Siebert, K., Hofmann, K. P., and Kreutz, W. (1978). Two distinct rhodopsin molecules within the disc membrane of vertebrate rod outer segments. Biochim. Biophys. Acta 503,450461. Hubbard, R., and Kropf, A. (1958). The action of light on rhodopsin. Proc. Natl. Acad. Sci. USA 44, 130-139. Hubbard, iodopsin.

R., and Kropf, A. (1959). Chicken Nature 783, 448-450.

lumi-

and

meta-

Hubbard, R., Bounds, D.,andYoshizawa,T. (1965).Thechemistry ofvisual photoreception. Cold Spring Harbor Symp. Quant. Biol. 30, 301-315. Kibelbek, J., Mitchell, D. C., Beach, J. M., and Litman, B. J. (1991). Functional equivalenceof metarhodopsin II and theG?-activating form of photolyzed bovine rhodopsin. Biochemistry 30, 67616768. King, P.J., and Cutfreund, H. (1984). Kinetic studies on theform’a tion and decay of metarhodopsins from bovine retinas. Vision Res. 24, 1471-1475. Konig, B., Welte, W., and Hofmann, of rhodopsin and interaction with celles. FEBS Lett. 257, 163-166. Kuwata, O., Matsumoto, K., Shimura, of iodopsin, 272, 128-132.

K. P. (1989). Photoactivation transducin in detergent mi-

Imamoto, Y., Okano, T., Kokame, K., Kojima, D., H., Morodome,A., Fukada, Y., Shichida, Y., Yasuda, Y., and Yoshizawa, T. (1990). The primary structure a chicken red-sensitive cone pigment. FEBS,Lett.

Longstaff, C., Calhoun, R. D., and Rando, R. R. (1986). Deprotonation of the Schiff base of rhodopsin is obligate in the activation of the G protein. Proc. Natl. Acad. Sci. USA 83, 4209-4213. Mathews, R. G., Hubbard, R., Brown, Tautomeric forms of metarhodopsin. 240.

P. K., and Wald, G. (1963). J. Gen. Physiol. 47, 215-

Nathans, J. (1990a). Determinants of visual pigment absorbance: role of charged amino acids in the putative transmembrane segments. Biochemistry 29, 937-942. Nathans, J. (1990b). Determinants of visual pigment absorbance: identification of the retinylidene Schiff’s base counterion in bovine rhodopsin. Biochemistry 29, 9746-9752. Nathans, J., and Hogness, D. S. (1983). Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin. Cell 34, 807-814. Nathans, J., and Hogness, D. S. (1984). Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc. Natl. Acad. Sci. USA 87, 4851-4855. Nathans, J., Thomas, D., and Hogness, D. S. (1986). Molecular genetics of human color vision: the genes encoding blue, green, and red pigments. Science 232, 193-202. Nathans, J., Weitz, C. J., Agarwal, N., Nir, I., and Papermaster, D. S. (1989). Production of bovine rhodopsin by mammalian cell

lines expressing lar localization.

cloned cDNA: spectrophotometryand Vision Res. 29, 907-914.

subcellu-

Neitz, M., Neitz, J., and Jacobs, G. H. (1991). Spectral tuning of pigments underlying red-green color vision. Science 252, 971974. Ovchinnikov, Y. A., Abdulaev, N. G., Feigina, M. Y., Atramonov, I. D., Bogachuk, A. S., Zolotarev, A. S., Eganyan, E. R., and Kostetskii, P. V. (1983). Visual rhodopsin Ill: complete amino acid sequenceand topography in the membrane. Bioorg. Khim. 9,13311340. Papermaster, D. S., and Dreyer, W. J. (1974). Rhodopsin content in theouter segment membranes of bovine and frog retinal rods. Biochemistry 73, 2348-2444. Parkes, J. H., and Liebman, P. A. (1984). Temperature and pH dependence of the metarhodopsin I-metarhodopsin II kinetics and equilibria in bovine rod disk membrane suspensions. Biochemistry 23, 50545061. Radding, C. R., and Wald, G. (1956). Acid-base properties dopsin and opsin. J. Gen. Physiol. 30, 909-923.

of rho-

Sakmar, T. P., Franke, R. R., and Khorana, H. C. (1989). Clutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Proc. Natl. Acad. Sci. USA 86, 8309-8913. Schoenlein, R. W., Peteanu, L. A., Mathies, R. A., and Shank, C. V. (1991). The first step in vision: femtosecond isomerization of rhodopsin. Science 254, 412-415. Stewart, J. G., Baker, B. N., and Williams, T. P. (1975). Kinetic evidence for a conformational transition in rhodopsin. Nature 258, 89-90. Straume, M., Mitchell, D. C., Miller, J. L., and Litman, B. j. (1990). lnterconversionofmetarhodopsins I and Il:a branched photointermediate decay model. Biochemistry 29, 9135-9142. Stryer, L. (1986). Cyclic CMP cascade rosci. 9, 87-119.

of vision.

Annu.

Rev. Neu-

Takao, M., Yasui, A., and Tokunaga, F. (1988). Isolation and sequence determination of the chicken rhodopsin gene. Vision Res. 28, 471-480. Wald, C. (1968). The molecular 219, 800-807.

basis of visual excitation.

Wulff, V. J., Adams, R. G., Linschitz, H., and Kennedy, Effect of flash illumination on rhodopsin in solution. Acad. Sci. 74, 281-290.

Nature D. (1958). Ann. NY

Yokoyama, R., and Yokoyama, S. (1990). Convergent evolution of the red- and green-like visual pigment genes in fish, Astyanax fascia, and human. Proc. Natl. Acad. Sci. USA 87, 9315-9318. Zhukovsky, E. A., and Oprian, D. D. (1989). Effect of carboxylic acid side chains on the absorption maximum of visual pigments. Science 246, 928-930.