Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 8, pp. 1341-1358 © Pleiades Publishing, Ltd., 2026.

1341

REVIEW

Genetic and Molecular Basis

of Cone Opsin Phototransduction

Hanwen Cao

, Nora Puig-Puig

, Alejandro Cruz

, Karl-Wilhelm Koch

3,a

and Pere Garriga

1,b

Grup de Biotecnologia Molecular i Industrial, Centre de Biotecnologia Molecular,

Departament d’Enginyeria Química, Universitat Politècnica de Catalunya-Barcelona Tech,

08222 Terrassa, Catalonia, Spain

Grup de Biotecnologia Molecular i Industrial, Centre de Biotecnologia Molecular,

Departament d’Enginyeria Química, Universitat Politècnica de Catalunya-Barcelona Tech,

08028 Barcelona, Catalonia, Spain

Department of Neuroscience, Division of Biochemistry, Carl von Ossietzky Universität Oldenburg,

26111 Oldenburg, Germany

e-mail: karl.w.koch@uni-oldenburg.de

e-mail: pere.garriga@upc.edu

Received April 2, 2026

Revised April 2, 2026

Accepted April 29, 2026

Abstract— The vertebrate visual system contains a sophisticated physiological assembly of highly-specialized

proteins that mediate light stimuli to be processed by the brain. Two types of cells, the rod and cone cells,

are involved in detecting dim and bright light, respectively. The rod cell contains rhodopsin whose structure

and function has been extensively studied and the molecular interactions involving rhodopsin and the other

proteins of the phototransduction cascade have been dissected in great detail, although several mechanistic

features remain to be determined. The cone phototransduction process has been less studied, particularly

at the cone opsin structural level, and some conformational and mechanistic information has been inferred

from analogy to the rod system. In spite of this, current efforts of investigation are focused on the study

of the structure and function of cone opsins and other proteins of the cone phototransduction system.

In this review we cover the current knowledge on the genetic and molecular aspects of cone opsins and

the consequences of cone opsin mutations on the structure and function of these photoreceptor proteins

causing vision disorders ranging from mild color vision abnormalities to severe cone-mediated retinal de-

generation. A second part of the review is dedicated to the analysis of the molecular interactions involving

calcium-binding proteins (such as recoverin and guanylate cyclase-activating proteins) in zebrafish which

has emerged as a very useful model organism for the study of cone opsin phototransduction particularly

in the deactivation steps of the visual phototransduction cascade.

DOI: 10.1134/S0006297926601061

Keywords: cone opsin structure, color vision, retinal degeneration, calcium-binding proteins, zebrafish

* To whom correspondence should be addressed.

INTRODUCTION

Cone opsins constitute the primary molecular

foundation of human photopic vision, high-acuity

sight, and color perception. As specialized G  pro-

tein-coupled receptors (GPCRs) localized within the

cone outer segments, these photopigments covalently

bind the chromophore 11-cis-retinal to convert light

energy into biochemical signals  [1,  2]. In the human

visual system, cone opsins are classified into three

distinct types based on their spectral absorption max-

ima: long-wavelength-sensitive  (L) opsins (encoded

by OPN1LW), middle-wavelength-sensitive  (M) opsins

(encoded by OPN1MW), and short-wavelength-sensi-

tive  (S) opsins (encoded by OPN1SW), which corre-

spond to the perception of red, green, and blue light,

CAO et al.1342

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

respectively  [3,  4]. Compared to rhodopsin in rods,

these three cone opsins exhibit unique spectral sen-

sitivities and significantly faster activation and de-

activation kinetics, which are essential for resolving

rapid temporal changes in natural light environ-

ments [5, 6].

Compared to rhodopsin, which mediates scoto-

pic vision, our understanding of the structural, bio-

chemical, and physiological properties of cone opsins

remains relatively limited. However, increasing evi-

dence demonstrates that mutations in cone opsins

can give rise to a wide spectrum of inherited retinal

disorders  [7-9]. Notably, due to the high sequence ho-

mology between the OPN1LW and OPN1MW genes,

this genetic locus is particularly susceptible to rear-

rangements and mutations [4, 10, 11]. Consequently,

these genetic variations result in cone photoreceptor

dysfunction, which is reflected clinically in a wide

spectrum of phenotypes, ranging from mild color vi-

sion defects to severe, progressive cone diseases [8,

12-14].

Although increasing recognition of cone opsin-as-

sociated disorders, a systematic overview of their ge-

netic organization and mutational landscape remains

important for understanding their clinical presen-

tation. This review covers the genetic architecture

and molecular properties of cone opsin subtypes, the

structural basis of cone opsin function, and the conse-

quences of disease-associated mutations ranging from

mild color vision deficiencies to severe cone-mediated

retinal degeneration, along with current therapeutic

approaches. We also address the molecular interac-

tions involving calcium-binding proteins in zebrafish

as a model organism for studying the deactivation

steps of the cone phototransduction cascade.

CONE OPSIN SUBTYPES

AND GENETIC ORGANIZATION

The vertebrate retina typically harbors two class-

es of photoreceptors: rods, which mediate dim-light

(scotopic) vision, and cones, which are responsible

for daylight (photopic) vision and color discrimina-

tion  [15]. At the heart of cone cell function are vi-

sual pigments, specialized opsins embedded within

the lipid bilayer of the outer segment membrane

discs  [16]. These pigments are characterized by dis-

tinct spectral sensitivities, providing the molecular

framework for color perception [2].

Human color vision is fundamentally trichromat-

ic, relying on the coordinated activity of three spec-

trally distinct cone populations, each defined by the

specific absorption maximum (λ

max

) of its expressed

opsin. Short-wavelength-sensitive  (S) cones express

S-opsin, with a λ

max

of approximately 419.0  ±  3.6  nm,

mediating blue color perception. Middle-wave-

length-sensitive  (M) cones express M-opsin, with a λ

max

of 530.8  ±  3.5  nm, corresponding to the green region

of the visible spectrum. Long-wavelength-sensitive  (L)

cones express L-opsin, with a peak absorption at

558.4  ±  5.2nm, mediating red color perception  [2,  17].

The genetic architecture encoding these phot-

opigments is characterized by unique chromosomal

distributions and structural complexities. In contrast

to the autosomal nature of the OPN1SW gene (located

at 7q32.1), the OPN1MW and OPN1LW genes are sit-

uated on the X chromosome (Xq28), where they are

uniquely organized in a head-to-tail tandem array [4,

18]. Notably, OPN1LW and OPN1MW are remarkably

similar at the sequence level, with over 98% nucle-

otide identity. This conservation is further illustrat-

ed at the protein level, where amino acid sequence

alignment reveals a high degree of similarity between

L-opsin and M-opsin, in contrast to the greater diver-

gence observed in S-opsin (Fig.  1). This close homol-

ogy renders the locus highly susceptible to unequal

homologous recombination  [4,  11,  18].

The canonical OPN1LW/OPN1MW gene array con-

sists of a single OPN1LW gene followed by one or

more copies of the OPN1MW gene [11,  19] (Fig.  2).

Crucially, transcriptional studies demonstrate that

only the two most proximal genes in the array con-

tribute to functional vision; specifically, the OPN1LW

gene and the immediately downstream OPN1MW

gene are transcriptionally active, whereas distal cop-

ies typically remain silent  [11]. The expression of this

gene cluster is governed by a Locus Control Region

(LCR) situated 3.1-3.7  kb upstream of the array  [20].

STRUCTURAL BASIS

OF CONE OPSIN FUNCTION

Cone opsins are characterized by their rapid

active-state decay and enhanced structural flexibil-

ity compared to rhodopsin  [21,  22]. This fundamen-

tal functional difference is because of the distinct

structural features of the protein. There are no re-

ported high-resolution atomic structures of the in-

active conformations of cone opsins and little de-

tailed structural information is available for these

cone pigments due to their high conformational in-

stability in purified systems. In spite of these lim-

itations, recent high-resolution cryo-electron micros-

copy (cryo-EM) and biophysical analyses of human

green cone opsin (GCO) reveal that its active state

(Meta  II) adopts a more “porous” and conformation-

ally relaxed structure, characterized by an expanded

chromophore-binding cavity, an enlarged water chan-

nel, and a potential “exit pathway” near the Schiff

base that facilitates rapid chromophore release  [22].

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Fig.  1. Multiple amino acid sequence alignment of cone opsins. Cone opsins protein sequences were aligned by ClustalW.

Conserved amino acids are highlighted in black, while residues with similar physicochemical properties are shaded in grey.

The alignment reveals a high degree of conservation between L-opsin and M-opsin across the sequence, whereas S-opsin

displays greater divergence.

Fig.  2. Structural organization and transcriptional regulation of the human OPN1LW/OPN1MW array. Both the OPN1LW

(L-cone opsin, red) and OPN1MW (M-cone opsin, green) genes consist of six exons. TheLCR, located approximately 3.1-3.7  kb

upstream, functions as a master regulatory element. It physically interacts with the promoter of only one opsin gene at

a time to initiate transcription. This exclusive, stochastic interaction ensures that only a single opsin type is expressed in

any given cone cell, while downstream copies of the array remain transcriptionally repressed.

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Fig.  3. Structural comparison of human GCO and rhodopsin active states. Structural comparison of human WT  GCO and

rhodopsin active states (in green and cyan, respectively) from a top-down with a zoom in the extracellular regions where

both opsins differ. Despite the overall structures of both active states are highly similar, the existing differences gather

around the orthosteric site (encircled in black), giving rise to distinctive features for each opsin. This cavity, which is re-

sponsible for accommodating the retinal, is flanked by two openings, referred to as Hole  A and  B (right and left panels,

respectively). Although both holes are larger in the case of human GCO, this size difference is especially remarkable for

Hole  A. These enlarged openings, along with larger cone orthosteric sites (bottom panel), contribute to their rapid decay

from the active state, because more water molecules can access and be accommodated into this cavity, favoring faster

retinal hydrolysis and release. Additionally, those sites contain strategically-placed polar residues that confer the specific

spectral features on each opsin. Regarding residue labels, the first amino acid (left) refers to GCO while the second one

refers to Rho (right). For the sake of better spatial orientation, retinal has been depicted in all panels (in orange and slate

gray for GCO and rhodopsin, respectively). The corresponding structure to the active-state human WT GCO was retrieved

from PDB code 9YDA [22], whereas that for the active-state human WT rhodopsin was generated with AlphaFold 3.

Structural comparison between GCO and rhodopsin

active states reveals that these differences are con-

centrated around the orthosteric site. Two distinct

openings, Hole  A and Hole  B, are notably enlarged in

GCO relative to rhodopsin, with Hole  A showing the

most pronounced difference (Fig.  3).

At the molecular level, a conserved anionic resi-

due E102, located beneath the Schiff base in red and

green cone opsins, plays a key role in modulating

active-state decay  [22]. Kinetic analyses indicate that

the accelerated Schiff base hydrolysis in GCO aris-

es primarily from a reduced entropic barrier rather

than differences in transition-state enthalpy. While

the transition-state enthalpies (ΔH

‡

) are compara-

ble, significant differences in transition-state entropy

(ΔS

‡

) are observed, suggesting that E102 promotes hy-

drolysis through entropic effects rather than direct

chemical catalysis. Consistently, neutralization of this

residue (GCO_E102Q) slows active-state decay and en-

hances G protein activation, highlighting its function-

al importance [22].

These structural and kinetic features are support-

ed by spectroscopic and biochemical studies demon-

strating that cone opsins exhibit faster Meta  II decay

and a greater propensity for spontaneous chromo-

phore dissociation compared to rhodopsin, reflect-

ing increased conformational dynamics within the

ligand-binding pocket. This intrinsic flexibility, while

associated with reduced pigment stability and fast-

er spontaneous chromophore release, underlies the

diverse conformational behaviors and regeneration

mechanisms observed across cone opsin subtypes

[21,  23]. For example, human red and green cone

opsins adopt distinct transient conformations during

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chromophore regeneration, underscoring the dynam-

ic nature of their binding pockets  [23]. In blue cone

opsin, this flexibility enables more complex regenera-

tion mechanisms involving secondary retinal binding

with analog specificity  [24].

From a physiological perspective, these seem-

ingly “unstable” structural features represent adap-

tive specializations for photopic vision. By promoting

rapid chromophore release and accelerating receptor

reset, they allow cone opsins to sustain high tempo-

ral resolution and prevent signal saturation under

bright–light conditions  [21,  22].

CLASSES OF CONE OPSIN MUTATIONS

The molecular landscape of cone-mediated vision

loss is uniquely shaped by the genomic architecture

of the visual pigment genes. At the Xq28 locus, the

OPN1LW and OPN1MW genes are organized in a

head-to-tail array. Due to this tandem arrangement

and the aforementioned high sequence homology, the

locus is particularly prone to unequal homologous re-

combination events during meiosis. In contrast, the

OPN1SW gene encoding the S-opsin is located on

the autosome (chromosome  7) and lacks this tandem

complexity, making it primarily susceptible to specif-

ic missense or splicing mutations rather than struc-

tural rearrangements. The resulting genetic instabil-

ity, along with various point mutations, manifests

as a diverse spectrum of cone-mediated vision loss.

The structural positions of representative pathogenic

mutations across L/M-opsin and S-opsin are illustrat-

ed in Fig.  4.

Protein misfolding and cytotoxic degenera-

tion. The most prevalent missense mutations, such

as C203R (Fig.  4a), fall into this category. The Cys203

residue is essential for forming a highly conserved

disulfide bond linking transmembrane helix  III and

extracellular loop  2 (ECL2)  [26]. The substitution of

Cysteine with Arginine disrupts this bond, leading

to misfolded opsin proteins that are retained in the

endoplasmic reticulum (ER). The resulting ER stress

triggers the Unfolded Protein Response (UPR), lead-

ing to progressive cone degeneration, characteristic

of BCM and Cone Dystrophy [27]. Other notable mis-

folding mutations in the L/M cluster include N94K

and W177R (Fig.  4a) [28,  29]. Similarly, misfolding is

a primary disease mechanism for the OPN1SW. In the

OPN1SW L56P variant, the introduction of a proline

is predicted to disrupt the TM1 helix, while other

variants such as G79R and P264S (Fig.  4b) are thought

to impair protein stability or local helical structure

[30,  31]. Like their L/M counterparts, these defective

S-opsins are retained in the endoplasmic reticulum

and fail to traffic to the outer segments, leading to

cellular stress and likely contributing to progressive

S-cone degeneration  [30].

Impaired pigment function and phototrans-

duction. Certain cone opsin mutations allow for

relatively stable protein folding but impair the pho-

tochemical or signaling cycle of the photopigment.

For instance, variants such as P307L (Fig.  4a) alter

the 11-cis-retinal binding pocket, disrupting the for-

mation of a functional pigment  [32]. Mutations like

R330Q and G338E (Fig.  4a) interfere with arrestin

binding, which prevents the proper quenching of the

activated state and consequently alters phototrans-

duction kinetics  [32]. In S-cone, a similar function-

al impairment is observed with the OPN1SW T190I

variant (Fig.  4b). Unlike misfolding mutations, T190I

allows for proper protein trafficking to the outer seg-

ments but alters the amino acid environment near

the chromophore-binding pocket, leading to profound

abnormalities in S-cone spectral sensitivity and sig-

naling  [31]. Clinical manifestations of these function-

al impairments typically range from stationary color

vision deficiencies, such as dichromacy and BCM, to

autosomal dominant tritanopia in the case of S-cone

variants.

Genomic deletions and null phenotypes. At the

severe end of the molecular spectrum are genetic

alterations that abolish functional opsin expression,

creating “null alleles”. In the OPN1LW/OPN1MW clus-

ter, this frequently occurs via large-scale genomic de-

letions, such as the loss of the LCR, which silences

transcription of the entire gene array  [33]. Similarly,

nonsense mutations like R247X (Fig.  4a) yield truncat-

ed, non-functional proteins  [34,  35]. Beyond DNA-lev-

el deletions, post-transcriptional splicing defects also

drive null phenotypes. Specific exon  3 haplotypes in

the OPN1LW/OPN1MW genes, as well as intron  3 nu-

cleotide deletions in the autosomal OPN1SW gene,

severely disrupt pre-mRNA splicing [36,  37]. This re-

sults in haploinsufficiency and a dramatic failure to

synthesize functional photopigments. Clinically, these

null alleles typically manifest as stationary color vi-

sion deficiencies, such as blue cone monochromacy

(BCM) or autosomal dominant tritanopia  [37,  38].

By avoiding the cytotoxic ER stress associated with

misfolding, these null-allele cones escape acute pro-

teotoxicity. However, they may still undergo second-

ary atrophy from chronic disuse and the loss of pho-

totransduction-mediated support  [39].

Recent studies suggest that some cone opsin mu-

tations cannot be readily assigned to a single patho-

genic category, highlighting the mechanistic hetero-

geneity underlying cone opsin-associated disorders.

For example, K82E (Fig.  4a) traffics predominantly to

cone outer segments and retains partial phototrans-

duction capacity, despite still reducing cone viabili-

ty[40]. Incontrast, P187S (Fig.  4a) is barely detectable

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Fig. 4. Structural localization of disease-associated mutations in cone opsins. Two-dimensional topological diagrams illustrate

the transmembrane organization of (a)  M-opsin and (b)  S-opsin (adapted from  [25]). Each circle represents a single amino

acid residue. Pathogenic mutations associated with color vision deficiencies or cone dystrophies are highlighted in red and

indicated by blue text boxes. In (a), the M-opsin sequence is shown as a representative of the L/M-opsin subfamily, the

mapped variants include K82E, N94K, W177R, P187S, C203R, R247X, M273K, P307L, R330Q, and G338E. In(b), the highlight-

ed S-opsin mutations are L56P, G79R, T190I, and P264S. The spatial distribution of these variants highlights the structural

diversity of pathogenic mechanisms across different domains of the cone opsin protein.

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by western blot or immunohistochemistry, likely due

to rapid proteasomal degradation of the mutant pro-

tein[40]. M273K (Fig.  4a) exhibits features of both de-

fective chromophore incorporation and intracellular

retention, suggesting overlapping defects in pigment

formation and protein trafficking  [40].

CLINICAL MANIFESTATIONS

AND THERAPEUTIC APPROACHES

Genotype-phenotype correlations and disease

entities. Mutations in cone opsin genes give rise to a

broad spectrum of clinical phenotypes, ranging from

isolated color vision deficiencies to severe progres-

sive cone degeneration. The clinical outcome largely

depends on the type of genetic alteration and its im-

pact on protein function and gene expression.

Anomalous trichromacy. Anomalous trichromacy

is the most common congenital color vision deficiency,

characterized by the presence of all three cone types

where one photopigment exhibits a shifted spectral

sensitivity  [41]. It primarily arises from non-allelic

homologous recombination between OPN1LW and

OPN1MW genes, creating hybrid genes with altered

absorption peaks  [42,  43].

Protanomaly and Deuteranomaly: These X-linked

conditions involve a spectral shift of the L-cone toward

the M-spectrum or the M-cone toward the L-spectrum,

respectively, leading to a “compressed” color space

and impaired red-green discrimination  [42].

Tritanomaly: Arare autosomal dominant condition

caused by mutations in the OPN1SW gene, affecting

blue-yellow perception [43]. Despite functional deficits,

adaptive optics imaging often reveals a structurally in-

tact cone mosaic in these patients, distinguishing them

from more degenerative phenotypes[41].

Dichromacy. Dichromacy involves the total func-

tional loss of one cone photoreceptor class, reducing

trichromatic vision to a two-channel system.

Protanopia and Deuteranopia: Resulting from the

complete absence of functional L-cones or M-cones,

these X-linked recessive disorders lead to severe red-

green color blindness. Their spectral sensitivity is

characterized by a “neutral point” where a specific

wavelength is perceived as achromatic, a phenome-

non that distinguishes them from trichromats  [44,  45].

Tritanopia: An  autosomal dominant disorder

caused by the loss of S-cone function. Research con-

firms that while the L- and M-cone pathways are op-

erational, the absence of S-cone input disrupts the

blue-yellow color dimension, often shifting the spec-

tral saturation curves as measured in psychophysical

tests  [44,  46].

Monochromacy. Monochromacy is the most se-

vere color vision deficiency, occurring when at least

two cone classes fail. Unlike dichromats, monochro-

mats rely on a single cone type or exclusively on

rods, leading to a total inability to perceive hues

and significant deficits in visual acuity and light ad-

aptation. This category primarily includes Blue Cone

Monochromacy (BCM), which retains S-cone function,

and Achromatopsia (ACHM), where all cone signaling

is absent.

Blue Cone Monochromacy (BCM): BCM is primar-

ily caused by the deletion of the Locus Control Re-

gion (LCR) or by intergenic recombination events that

result in deleterious point mutations, such as C203R

(which disrupts a critical disulfide bond) or R247X

[33,  34]. The clinical manifestations of BCM include

severe color vision impairment, nystagmus, photo-

phobia, and high myopia  [32].

Achromatopsia (ACHM): ACHM is an autosomal

recessive disorder caused by mutations in genes essen-

tial for the cone phototransduction cascade, most no-

tably CNGA3 and CNGB3 (accounting for 75% of cases),

alongside GNAT2, PDE6C, PDE6H, and ATF6  [15,  47].

Unlike BCM, which preserves S-cone function, ACHM

patients suffer from a complete loss of signaling in all

three cone types (L, M, and S) due to the functional

failure of CNG channels or related transduction pro-

teins  [48]. Clinical manifestations present from birth

and including total color blindness, significantly re-

duced visual acuity, severe photophobia, and pendular

nystagmus, with potential slow retinal degeneration of

the macular area observed over time  [47,  49].

Current and emerging therapeutic approach-

es. Gene augmentation utilizing adeno-associated vi-

rus (AAV) vectors represents a promising therapeutic

strategy for cone opsin-associated disorders. Preclin-

ical studies in animal models have demonstrated

that delivery of functional opsin genes can restore

cone-mediated visual function and, in some cases, im-

prove cone structural integrity. The feasibility of this

approach was notably demonstrated by Gene thera-

py for red-green color blindness in adult primates,

where AAV-mediated delivery of L-opsin conferred

trichromatic color vision in adult dichromatic non-hu-

man primates  [50]. Importantly, this approach is not

restricted to simple loss-of-function contexts. In mod-

els carrying the prevalent C203R missense mutation,

which causes cytotoxic protein misfolding, AAV-me-

diated delivery of wild-type opsin has been shown

to outcompete mutant proteins, partially rescuing

both cone morphology and photopic function  [27].

Furthermore, the broad utility of cone-targeted AAV

vectors is evidenced by their efficacy in other inher-

ited cone degenerations, such as achromatopsia  [51].

Crucially for clinical translation, high-resolution hu-

man retinal imaging confirms that a substantial pop-

ulation of foveal cones remains structurally intact

despite opsin defects, providing a vital therapeutic

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

window for gene intervention even in advanced dis-

ease stages [52].

Despite these advances, gene augmentation alone

may not be sufficient for all classes of cone opsin

mutations. In particular, mutations associated with se-

vere protein misfolding, or dominant-negative effects

are unlikely to be fully corrected by simply increas-

ing wild-type opsin expression, as the underlying pro-

teotoxicity remains unresolved. In such cases, ther-

apeutic strategies must directly address the specific

molecular defects. Promising experimental approach-

es include stabilizing misfolded proteins via pharma-

cological chaperones  [53] and selectively suppressing

mutant alleles through gene-specific silencing or ge-

nome editing  [54]. Although these advanced methods

are currently in the early stages of development for

cone opsin-specific disorders, their successful appli-

cation in other dominantly inherited retinal degen-

erations highlights the potential for a more refined,

mechanism-based therapeutic framework.

The evolving landscape of cone opsin therapeu-

tics underscores that treatment efficacy is fundamen-

tally dictated by the specific mutational mechanism.

AAV-mediated gene augmentation provides a highly

effective solution for pure loss-of-function alleles and

early-stage interventions. However, effectively manag-

ing more complex pathogenic variants will necessitate

the ongoing development of tailored, mutation-specif-

ic strategies to fully combat underlying proteotoxicity.

In spite of the current efforts to unravel the phe-

notype-genotype linkage, that underlies vision color

deficiencies and cone retinal degeneration, for the de-

velopment of effective therapeutic approaches, there

is a need to decipher the precise molecular interac-

tions involved in cone opsin phototransduction and

their differences with the rod opsin system which

has been much more studied. It is of particular rele-

vance to elucidate cone opsin inactive structures and

the molecular mechanisms of signal transduction in

cones, particularly for the photoreceptor deactiva-

tion process involving regulatory proteins, enzymes

and calcium binding proteins, such as recoverin and

guanylate cyclase-activating proteins. In the follow-

ing sections we will describe recent developments in

the study of these molecular interactions in zebrafish

that has emerged as an excellent model organism for

the study of cone visual phototransduction.

PHOTORESPONSES FROMTELEOST FISH

ASMODEL ORGANISMS

Teleost fish, mainly carp (Cyprinus carpio) and

zebrafish (Danio rerio) were established in recent de-

cades as model organisms in vision research [55-58].

In particular zebrafish draw attention for the study

of cone photoreceptors and cone phototransduction.

Several reasons favor zebrafish as model organism

for the study of cone vision. For example, unlike the

rodent retina, the zebrafish retina harbors mainly

cone cells (60% in total), which are double cones

(long-wavelength sensitive, red and green), long sin-

gle cones (short-wavelength sensitive, blue) and short

single cones (UV-sensitive, violet) in addition to rods

(upper part of Fig.  5). Electroretinography (ERG) re-

cordings as well as single-cell suction electrode re-

cordings can be applied to monitor photoresponses

under different illumination protocols[59-62]. A com-

parative analysis of flash response sensitivity revealed

that rods are 40-220-fold more sensitive than cones,

and green-sensitive cones exhibit higher sensitivity

than red-sensitive ones. But more surprising in this

study were the smaller flash responses in rods com-

pared to cones  [62]. Earlier recordings from red-sen-

sitive carp cones are in agreement with a lower light

sensitivity in cones than in rods (~10

-fold), and the

lower sensitivity could be based on more rapid pig-

ment phosphorylation by a higher opsin kinase ac-

tivity  [63].

Further, morpholino based gene-targeted knock

down approaches and more recently CRISPR/Cas ge-

nome editing allow the combination of physiology,

genetic analysis and vision based behavioral stud-

ies [64-66].

EXPRESSION OF CONE SPECIFIC

GENES ANDPROTEINS INVOLVED

INPHOTOTRANSDUCTION

Gene and genome duplication in zebrafish has

led to a complex presence and expression of paralog

genes  [67]. Nearly all key proteins of the phototrans-

duction pathway are present in several paralogs

[68-70]. For example, there are one UV opsin, one

blue opsin, four green opsin, two red opsin and one

rod opsin proteins encoded in the zebrafish genome

(for a summarizing table including other phototrans-

duction proteins see  [71]). Expression and localization

of these key phototransduction proteins have been re-

ported showing that the basic steps of phototransduc-

tion (Fig.  6) operate in zebrafish cone cells similar to

the well-established signaling pathway in vertebrate

rod outer segments. However, the complexity of the

expression profiles in the different cone photorecep-

tor cell types and their functions have not been ful-

ly explored. This leaves open questions like how do

differences in paralog protein function account for

differences in photoresponse kinetics and adaptation.

Or, do genes exhibit equal expression profiles during

development from the larval to the adult state? Did

some paralog proteins develop unknown functions

CONE OPSIN PHOTOTRANSDUCTION 1349

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Fig.  5. Rod and cones in the teleost (in particular) zebrafish retina. The upper part shows the different cone cell types

indicating the outer segment (localization of phototransduction), inner segment and cell body, and the synaptic terminal.

The lower part lists the expression of zGCAP paralogs. Yellow background marks IC

values at 23 to 35 nM free Ca

-con-

centration, green background a range from 180 to 520 nM (see also Fig. 7).

Fig.  6. Photoexcitation and Ca

-dependent feedback loops. The main steps of the phototransduction cascade are briefly

displayed: rhodopsin (Rh) is illuminated leading to Meta-rhodopsin  II (Rh*) that activates the G protein transducin by

catalyzing the GDP to GTP exchange at the α-subunit. The GTP-bound transducin α-subunit activates Phosphodiesterase  6

(PDE6), which hydrolyzes cGMP to 5′-GMP. In the dark state of the cell, cGMP binds to the CNG-channel leading to open-

ing of the channel and an influx of mainly Na

and to a lower extent of Ca

. The latter is extruded via a Na

/Ca

, K

exchanger, which balances a cytoplasmic Ca

-concentration in the dark around 500nM depending on the species. Closure

of CNG-channels by hydrolysis of cGMP stops Ca

-entry, but not its extrusion leading to net decrease of cytoplasmic Ca

Changes in cytoplasmic Ca

are sensed by Ca

-binding proteins such as recoverin (Rec), GCAPs and calmodulin (CaM),

which triggers three negative feedback loops: to deactivate rhodopsin by facilitating phosphorylation viaGRK1, to increase

the synthesis rate of guanylate cyclases (GC) and to modulate the affinity of the CNG-channel, when CaM dissociates from

the channel at low Ca

-concentration.

CAO et al.1350

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

by interacting with different targets? We will discuss

these points in the framework of the Ca

-feedback

regulation of phototransduction.

-FEEDBACK INVOLVING

-SENSOR PROTEINS

Deactivation of the steps in photoexcitation

and returning the photoreceptor to the dark or a

light-adapted state involves three Ca

-dependent

feedback regulatory mechanisms that were first dis-

covered in mammalian rod cells involving Ca

-sensor

proteins (Fig.  6). Rhodopsin kinase is regulated by re-

coverin  [72-75], membrane-bound guanylate cyclases

are under control of guanylate cyclase-activating pro-

teins  [76-78] and the cyclic nucleotide-gated (CNG)

channel is modulated by calmodulin or CNG-modulin

in zebrafish cones [79-81].

Light-activated rhodopsin is phosphorylated by

rhodopsin kinase (alternative name G  protein-coupled

receptor kinase  1, GRK1). This phosphorylation of il-

luminated rhodopsin and cone opsins by GRKs is a

critical step in the shut-off of the excitation pathway

in phototransduction and essential to return the cell

to the dark state. Inmost mammals, two GRK isoforms

have been identified, which are expressed in rods

(GRK1) and cones (GRK1 and GRK7) in a species-spe-

cific manner[82]. Inthezebrafish retina are four dif-

ferent isoforms of GRK (zGRK1a, zGRK1b, zGRK7a, and

zGRK7b corresponding to genes grk1a, grk1b, grk7a,

and grk7b, respectively, present [83, 84]). Recombinant

zGRK isoforms can phosphorylate bovine rhodopsin in

a light-dependent manner [84] confirming the critical

role zGRKs might also play in rod and cone photo-

response termination in zebrafish phototransduction.

Recoverin isoforms. The activity of rhodopsin

kinase is under control of a small Ca

-sensor pro-

tein named recoverin. The seminal work of the late

Pavel P.Philippov and his group was centered on the

characterization of mammalian recoverin, its impact

on GRK1 regulation and its role in cancer-associat-

ed retinopathy [73, 75, 85-87]. Biochemical properties

and structural features of mammalian recoverin have

been addressed in detail in many reviews and will

not be covered in this contribution. However, they

serve as benchmarks when investigating recoverin

ortholog features.

The zebrafish retina expresses four zebrafish re-

coverin variants (zRec1a, zRec2a, zRec1b, zRec2b cor-

responding to genes rcv1a, rcv1b, rcv2a, and rcv2b,

respectively). Initial characterization of zRec isoforms

showed their expression patterns in the adult retina

and a morpholino-based targeted gene knockdown

gave first hints to their in  vivo function, which in-

dicates target regulation of zebrafish specific GRK

isoforms and differences in Ca

-sensitivities of zRec

variants [83, 88].

However, a detailed expression analysis indicates

a more complex picture, since the expression level of

recoverin forms in the adult zebrafish retina is not

equal among zRec orthologs. Comparing zRec2b with

zRec1b yielded an up to 80-fold difference  [89] and

immunohistochemical labelling showed presence of

zRec1a throughout all rod cell substructures. In con-

trast, zGRK1 is prominently present in rod outer seg-

ments indicating a role as the main target of zRec1a.

The ortholog zRec2b has a more restricted expres-

sion to double cones co-localizing with zGRKs 1b, 7a,

and 7b [88, 89], but the four different zGRKs show

nearly equal expression levels [89]. The early devel-

opment of the visual system in zebrafish allows re-

cordings of electroretinograms(ERG) in zebrafish lar-

vae harboring knockdowns of zGRK1b and zGRK7a.

Recordings show a delay of photoresponse recovery

(Table  1) in agreement with a role in efficient opsin

deactivation [83, 90].

The flash response data of zRec2a and zRec2b

knockdowns in Table  1 show operation of zRec2a

and zRec2b under different light regimes pointing

to different Ca

-sensitive properties. The differential

expression pattern of zRec orthologs could further in-

dicate a function beyond regulation of zGRK. Changes

in expression levels of key proteins including zRec

orthologs during development seem to be under cir-

cadian control [57].

The Ca

-sensor properties of recoverin are key

for understanding its function, which is also apparent

for zRec orthologs. Elbers et al. [91] investigated the

differential response properties of zRec to oscillating

Table  1. Summary of the results reported by Zanget  al.

(2015) showing the consequences for cone recovery

after morpholino-based targeted gene knockdown

Knockdown Effect on cone flash response

recovery

zRec1a no effect

zRec2a quicker bright flash response

recovery

zRec2b normal response kinetics,

acceleration under dim light

zRec1a + zRec2a quicker bright flash response

recovery

zRec2a + zRec2b acceleration of response decay

under dim flash conditions

zGRK7a delay of recovery

zGRK7a + zRec2a no additional reduction or delay

CONE OPSIN PHOTOTRANSDUCTION 1351

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

changes in cytoplasmic Ca

-concentration, namely to

investigate possible Ca

-myristoyl switches, to deter-

mine their Ca

-binding properties and conformation-

al dynamics.

Due to the presence of the myristoylation con-

sensus sequence in the zRec amino acid sequences,

these proteins can have a myristoyl group attached

and could undergo a Ca

-myristoyl switch like bo-

vine recoverin. However, only zRec1a, and to a lesser

extent zRec2a, bound to membranes in a Ca

-depen-

dent manner. A clear Ca

-myristoyl switch mecha-

nism [92] was not observed for other myristoylated

zRec forms. Surprisingly, the non-myristoylated zRec

forms except zRec2b bound stronger to the membrane

in their Ca

-free state. Incontrast, zRec2b showed no

difference between its Ca

-free and Ca

-bound state.

The two functional EF-hands in bovine recoverin dis-

play sites of high and low affinity [85, 87, 92], which

was also observed for zRec forms, but all non-myris-

toylated zRec forms harbor lower affinities for Ca

, a

significant difference to bovine recoverin [91].

Guanylate cyclase-activating proteins. A key

enzyme in vertebrate phototransduction is a mem-

brane-bound guanylate cyclase (GC), which synthesiz-

es the second messenger of photoexcitation, guanosine

3′,5′-cyclic monophosphate (cGMP). Vertebrate GCs are

regulated by Ca

-sensor proteins named guanylate

cyclase-activating proteins (GCAPs) that detect chang-

es in cytoplasmic Ca

via their EF-hand Ca

-binding

motifs (Fig.  6). GCAPs share structural features with

bovine and other recoverin orthologs, and belong to

the family of neuronal calcium sensor (NCS) proteins.

They are expressed in different isoforms from two

or three in mammals to six or eight in teleost fish

[93-95]. A common feature of GCAPs is a switch in

conformation triggered by binding or dissociation of

. GC activities are high, when Ca

is low and at

least one Mg

-ion is bound in exchange for Ca

[96].

GC activities are low or even suppressed at saturat-

ing Ca

-concentration. Thus, a rearrangement of the

whole GC-GCAP complex increases or decreases GC

activities thereby controlling the cytoplasmic cGMP

concentration [97-101]. Resynthesis of cGMP after il-

lumination and cGMP depletion accelerates photore-

sponse recovery and oscillating cytoplasmic Ca

-con-

centrations in Ca

-feedback loops contribute to light

adaptation.

GCAPs form a complex with the target GC at

low and high Ca

-concentration, but apparent af-

finity constants of the interaction process are in the

lower micromolar to submicromolar range indicating

a transitory and flexible complex formation [102].

GCAPs are expressed in different isoforms from two

or three in mammals to six or eight in teleost fish

[94, 95, 103-105]. Detailed biochemical characteriza-

tion of GCAP properties showed that GCAPs differ

in their Ca

-sensitivity, Ca

-binding properties and

target regulatory features [96, 98, 106], which is in

agreement with electrophysiological recordings on

transgenic mice [107-109]. Thus, GCAPs activate the

target GC in a sequential order depending on the

actual cytoplasmic Ca

-concentration in the rod or

cone cell [98, 110, 111]. For example, mammalian

GCAP1 is active at higher Ca

-concentration than

GCAP2. When Ca

-decreases in the cell after illumi-

nation, GCAP1 will first loose its bound Ca

and turn

into an activator before GCAP2 would step into this

-feedback loop. This concept of GCAP regulatory

modes has been dubbed “Ca

-relay” or “recruitment

model” of GCAP action and is confirmed by computa-

tional approaches simulating excitation and recovery

of the photoresponses [111, 112].

The activation profiles and Ca

-dependent reg-

ulatory features of mammalian GCAPs are found in

rods and cones, although the expression of GCAP1

appears higher in mice cones. However, GCAP2 regu-

lates cGMP synthesis, when mice cones are deficient

of GCAP1 [113]. A more complex pictures emerges in

the zebrafish retina, which expresses six zebrafish

GCAPs paralogs (abbreviated zGCAPs) showing differ-

ences in spatial-temporal expression profiles in the

retina [94, 95, 104, 105, 114]. Rods and short single

cones express zGCAP1 and zGCAP2 and the isoforms

zGCAP3, zGCAP4, zGCAP5, and zGCAP7 are expressed

in all cone cells (double cones, long and short single

cones), at different levels showing high expression

for zGCAP3 and zGCAP4 and lower levels for zGCAP5

and 7 (Fig.  5, lower part). One remarkable feature of

zGCAPs is however that each form has distinct acti-

vation and Ca

-sensing profiles. Maximal activities of

zGCAP1, 5 and 7 are rather low (x-fold activation ex-

pressed as GC

max

−GC

min

divided by GC

min

is 1.5-3-fold),

but zGCAP2, 3 and 4 are strong activators having an

x-fold activation between 6 and13[103]. Furthermore,

the Ca

-sensitive regulation of GC targets exhibits

differences in IC

values (corresponding to [Ca

] at

which activation is half maximal), which are either

around 30  nM or around 400  nM Ca

  [103]. ZGCAPs

can therefore be classified into two groups according

to the Ca

-sensitivity. Each cone cell type contains a

combination of strong and weak GC activators (Fig.  6)

[115] and all cone types express also a combination of

zGCAPs having different IC

values (Fig.  7). Among all

six zGCAP paralogs zGCAP5 has unique properties as

it shows a very low degree of x-fold activation [103],

and contains two additional cysteine residues that can

ligate Fe

in a dimeric complex  [116] constituting an

inactive state  [117]. Therefore, it is unclear whether

zGCAP5 is a regulator of GC activity or targets to oth-

er proteins in cone cells. In summary, the findings

about zGCAPs indicate that they could also operate in

a sequential mode (Ca

-relay or recruitment model)

CAO et al.1352

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Fig.  7. Control of GC activity by zGCAP orthologs as a func-

tion of free Ca

-concentration. The yellow box highlights

the operation range of zGCAPs with a low IC

value, the

green box indicates a range at higher IC

values. Changes

of cytoplasmic Ca

from the dark to the illuminated state

of the cell is illustrated by the black-to-gray bar.

by detecting gradual changes in cytoplasmic Ca

their mammalian orthologs. It remains unsolved how

the differential expression and Ca

-sensitive regula-

tion of zGCAPs is involved in fine-tuning the cone light

responses at different wavelengths and how each pa-

ralog contributes to the Ca

-dependent mechanisms

of light adaptation.

Sensory guanylate cyclases in zebrafish reti-

na. The zebrafish genome harbors the genes gucy2f,

gc2, and gc3 encoding three membrane-bound sen-

sory GCs, namely zGC1, zGC2 and zGC3, respectively.

Transcription analysis of these genes revealed cone

specific expression of zGC3 in short single cones, long

single cones, and double cones, which was different

from the transcription of zGC1 and 2 in rods and

short single cones [95]. Gene-targeting to knockdown

the gucy2f gene by a morpholino based approach

caused shortening of outer segments. Zebrafish larvae

displayed impaired visual acuity detected by the opto-

motor response assay six days post fertilization [118].

The optomotor response in combination with the op-

tokinetic response served in a behavioral screening

assay of mutagenized zebrafishes. Muto et al. [65]

identified a visually impaired mutant named zatoichi

that has defects in the gc3 gene. The critical role of

the gc3 gene encoding zGC3 was further demonstrat-

ed by its immunolocalization in double cone, short

single and long single cone outer segments probed

by a specific anti zGC3 antibody [105]. These stud-

ies show the importance of zGCs for the processing

of visual information, but further work is needed

to characterize protein function on the cellular and

molecular level involving functional heterologous

expression, reconstitution experiments with zGCAP

forms and the characterization of their impact on

photoresponse recovery.

CONCLUSIONS

AND FUTURE PERSPECTIVES

Recent advances in cone opsin structural elucida-

tion have provided novel insights into the molecular

determinants of cone opsin photoactivation and the

initial steps of cone phototransduction. The main de-

velopments in this field have been covered in the first

part of this review. Furthermore, the study of model

systems has enabled the unraveling of functional fea-

tures of downstream effectors; specifically, the use of

zebrafish has provided key insights into the structure

of cone guanylate cyclases (and guanylate cyclase as-

sociated proteins) and the Ca

-mediated feedback

mechanisms in which they are involved.

Despite these significant strides, the cone pho-

totransduction system is far from being fully under-

stood, with many current models still relying heavily

on our deeper knowledge of the rod system. Future

research must focus on gaining a more detailed, in-

dependent picture of vertebrate cone phototransduc-

tion. Achieving this will require a multidisciplinary

approach – integrating protein structural determina-

tion, biochemistry, and functional analysis – to final-

ly provide a comprehensive description of the verte-

brate visual system as a whole.

Acknowledgments

The members of the two collaborating laboratories

(Koch and Garriga teams) are gratefully acknowledged

for contributing to the research in visual phototrans-

duction that has led to this review article.

Contributions

Hanwen Cao drafted the main manuscript, integrated

the comprehensive content, and performed the final

revisions. Nora Puig-Puig contributed to the conceptu-

alization of the review, drafted specific sections, and

assisted in the critical revision of the manuscript.

Alejandro Cruz generated the structural figures com-

paring the active states of human green cone opsin

and rhodopsin, and contributed to their interpreta-

tion. Karl-Wilhelm Koch drafted the section on cal-

cium-binding proteins and cone phototransduction

deactivation in zebrafish and provided overarching

supervision. PereGarriga provided overall project su-

pervision, critically reviewed, and audited the manu-

script. All authors have read and approved the final

version of the manuscript.

Funding

The experimental work in the laboratory of K.-W. Koch

was supported by grants from the Deutsche Forschungs-

gemeinschaft (KO948/15-2, project no. 322057463 and

GRK  1885/2; project no. 216581600). The work in the

laboratory of P.Garriga was supported by grants from

CONE OPSIN PHOTOTRANSDUCTION 1353

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Ministry of Science and Innovation, Spain (PID2019-

104817GB-I00), and from the Government of Catalonia

to Research Consolidated Groups (2021  SGR  00342).

H.  Cao is the recipient of a predoctoral grant from

CSC (Chinese Scholarship Council). N.  Puig-Puig is the

recipient of a predoctoral grant (FPI-UPC 2025) from

Universitat Politècnica de Catalunya – BarcelonaTech

(UPC-Banco de Santander).

Ethics approval and consent to participate

This review article does not contain any studies with

human participants or animals performed by any of

the authors.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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Proof Note. We acknowledge the recent publication of three cone opsins structural articles during the

preparation of our review. These studies have reported: (i) the atomic resolution dark-state structures of the

hGCO and hBCO (https://doi.org/10.1126/science.adz3624), (ii) the structure of the active cone opsins in com-

plex with Gi (https://doi.org/10.1126/science.adz8141), and (iii) the structural basis of spectral tuning in the

three cone opsins (https://doi.org/10.1126/science.adz3996). These three articles confirm previous experimental

observations and provide a more complete overall picture of the different spectral tuning, and activation/

deactivation mechanisms of cone opsins.