Gene therapy for achromatopsia

The present review summarizes the current status of achromatopsia (ACHM) gene therapy‐related research activities and provides an outlook for their clinical application. ACHM is an inherited eye disease characterized by a congenital absence of cone photoreceptor function. As a consequence, ACHM is associated with strongly impaired daylight vision, photophobia, nystagmus and a lack of color discrimination. Currently, six genes have been linked to ACHM. Up to 80% of the patients carry mutations in the genes CNGA3 and CNGB3 encoding the two subunits of the cone cyclic nucleotide‐gated channel. Various animal models of the disease have been established and their characterization has helped to increase our understanding of the pathophysiology associated with ACHM. With the advent of adeno‐associated virus vectors as valuable gene delivery tools for retinal photoreceptors, a number of promising gene supplementation therapy programs have been initiated. In recent years, huge progress has been made towards bringing a curative treatment for ACHM into clinics. The first clinical trials are ongoing or will be launched soon and are expected to contribute important data on the safety and efficacy of ACHM gene supplementation therapy.     

Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.     

A mutation in gene CNGA3 is associated with day blindness in sheep

Lambs with congenital day blindness show diminished cone function, which is characteristic of achromatopsia, a congenital disorder described in humans and dogs. To identify gene(s) associated with sheep day blindness, we investigated mutations in the CNGA3, CNGB3, and GNAT2 genes which have been associated with achromatopsia. Sequencing the coding regions of those genes from four affected and eight non-affected lambs showed that all affected lambs were homozygous for a mutation in the CNGA3 gene that changes amino acid R236 to a stop codon. By PCR-RFLP-based testing, homozygosity for the stop codon mutation was detected in another 19 affected lambs. Non-affected individuals (n = 386) were non-carriers or heterozygous for the mutation. While a selection program has been launched to eradicate the day blindness mutation from Improved Awassi flocks, a breeding nucleus of day-blind sheep has been established to serve as animal models for studying human achromatopsia.     

A Nonsense Mutation in PDE6H Causes Autosomal-Recessive Incomplete Achromatopsia

Achromatopsia (ACHM) is an autosomal-recessive retinal dystrophy characterized by color blindness, photophobia, nystagmus, and severely reduced visual acuity. Its prevalence has been estimated to about 1 in 30,000 individuals. Four genes, GNAT2, PDE6C, CNGA3, and CNGB3, have been implicated in ACHM, and all encode functional components of the phototransduction cascade in cone photoreceptors. Applying a functional-candidate-gene approach that focused on screening additional genes involved in this process in a cohort of 611 index cases with ACHM or other cone photoreceptor disorders, we detected a homozygous single base change (c.35C>G) resulting in a nonsense mutation (p.Ser12^∗) in PDE6H, encoding the inhibitory γ subunit of the cone photoreceptor cyclic guanosine monophosphate phosphodiesterase. The c.35C>G mutation was present in three individuals from two independent families with a clinical diagnosis of incomplete ACHM and preserved short-wavelength-sensitive cone function. Moreover, we show through immunohistochemical colocalization studies in mouse retina that Pde6h is evenly present in all retinal cone photoreceptors, a fact that had been under debate in the past. These findings add PDE6H to the set of genes involved in autosomal-recessive cone disorders and demonstrate the importance of the inhibitory γ subunit in cone phototransduction.     

Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitivity and pore properties of heteromeric channels

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to form by assembly of two different subunit types, CNGA3 and CNGB3. Recently, mutations in the gene encoding the CNGB3 subunit have been linked to achromatopsia in humans. Here we describe the functional consequences of two achromatopsia-associated mutations in human CNGB3 (hCNGB3). Co-expression in Xenopus oocytes of human CNGA3 (hCNGA3) subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels. In contrast, co-expression of a presumptive null mutation of hCNGB3 (T383f.s.Delta C) with hCNGA3 produced channels with properties indistinguishable from homomeric hCNGA3 channels. The effect of hCNGB3 S435F subunits on cell-surface expression of green fluorescent protein-tagged hCNGA3 subunits and of non-tagged hCNGA3 subunits on surface expression of green fluorescent protein-hCNGB3 S435F subunits were similar to those observed for wild type hCNGB3 subunits, suggesting that the mutation does not grossly disturb subunit assembly or plasma membrane targeting. The S435F mutation was also found to produce changes in the pore properties of the channel, including decreased single channel conductance and decreased sensitivity to block by l-cis-diltiazem. Overall, these results suggest that the functional properties of cone CNG channels may be altered in patients with the S435F mutation, providing evidence supporting the pathogenicity of this mutation in humans. Thus, achromatopsia may arise from a disturbance of cone CNG channel gating and permeation or from the absence of functional CNGB3 subunits.     

Homozygosity Mapping Reveals PDE6C Mutations in Patients with Early-Onset Cone Photoreceptor Disorders

Cone photoreceptor disorders form a clinical spectrum of diseases that include progressive cone dystrophy (CD) and complete and incomplete achromatopsia (ACHM). The underlying disease mechanisms of autosomal recessive (ar)CD are largely unknown. Our aim was to identify causative genes for these disorders by genome-wide homozygosity mapping. We investigated 75 ACHM, 97 arCD, and 20 early-onset arCD probands and excluded the involvement of known genes for ACHM and arCD. Subsequently, we performed high-resolution SNP analysis and identified large homozygous regions spanning the PDE6C gene in one sibling pair with early-onset arCD and one sibling pair with incomplete ACHM. The PDE6C gene encodes the cone α subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase, which converts cGMP to 5′-GMP, and thereby plays an essential role in cone phototransduction. Sequence analysis of the coding region of PDE6C revealed homozygous missense mutations (p.R29W, p.Y323N) in both sibling pairs. Sequence analysis of 104 probands with arCD and 10 probands with ACHM revealed compound heterozygous PDE6C mutations in three complete ACHM patients from two families. One patient had a frameshift mutation and a splice defect; the other two had a splice defect and a missense variant (p.M455V). Cross-sectional retinal imaging via optical coherence tomography revealed a more pronounced absence of cone photoreceptors in patients with ACHM compared to patients with early-onset arCD. Our findings identify PDE6C as a gene for cone photoreceptor disorders and show that arCD and ACHM constitute genetically and clinically overlapping phenotypes.     

Endoplasmic reticulum stress-associated cone photoreceptor degeneration in cyclic nucleotide-gated channel deficiency

Cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 account for >70% of all known cases of achromatopsia. Cones degenerate in achromatopsia patients and in CNGA3(-/-) and CNGB3(-/-) mice. This work investigates the molecular basis of cone degeneration in CNG channel deficiency. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we generated mouse lines with CNG channel deficiency on a cone-dominant background, i.e. CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) mice. The retinal phenotype and potential cell death pathways were examined by functional, biochemical, and immunohistochemical approaches. CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) mice showed impaired cone function, opsin mislocalization, and cone degeneration similar to that in the single knock-out mice. The endoplasmic reticulum stress marker proteins, including Grp78/Bip, phospho-eIF2α, phospho-IP(3)R, and CCAAT/enhancer-binding protein homologous protein, were elevated significantly in CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) retinas, compared with the age-matched (postnatal 30 days) Nrl(-/-) controls. Along with these, up-regulation of the cysteine protease calpains and cleavage of caspase-12 and caspase-7 were found in the channel-deficient retinas, suggesting an endoplasmic reticulum stress-associated apoptosis. In addition, we observed a nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G in CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) retinas, implying a mitochondrial insult in the endoplasmic reticulum stress-activated cell death process. Taken together, our findings suggest a crucial role of endoplasmic reticulum stress in cone degeneration associated with CNG channel deficiency.     

An intersubunit interaction regulates trafficking of rod cyclic nucleotide-gated channels and is disrupted in an inherited form of blindness

A mutation in a cyclic nucleotide-gated channel (CNGA1) is associated with retinitis pigmentosa (RP), a common, inherited eye disease. Expression of mutant (CNGA1-RP) homomeric channels in Xenopus oocytes revealed no measurable differences compared to wild-type CNGA1 homomers. As native retinal rod CNG channels comprise CNGA1 and CNGB1 subunits, we coexpressed CNGA1-RP and CNGB1. Surprisingly, this subunit combination did not produce detectable channels at the membrane surface. We show that the mechanism underlying this defect involves an intersubunit interaction between CNGA1 and CNGB1 that was not formed between CNGA1-RP and CNGB1 subunits. In the absence of this interaction, a short N-terminal region in CNGB1 prevented membrane expression. Thus, disruption of a regulatory interaction by mutation in CNGA1 exposed a region of CNGB1 that disrupted surface expression of heteromeric CNGA1-RP/CNGB1 channels, accounting for this instance of RP.     

Disease Progression in CNGA3 and CNGB3 Retinopathy; Characteristics of Slovenian Cohort and Proposed OCT Staging Based on Pooled Data from 126 Patients from 7 Studies

Achromatopsia has been proposed to be a morphologically predominately stable retinopathy with rare reports of progression of structural changes in the macula. A five-grade system of optical coherence tomography (OCT) features has been used for the classification of structural macular changes. However, their association with age remains questionable. We characterized the Slovenian cohort of 12 patients with pathogenic variants in CNGA3 or CNGB3 who had been followed up with OCT for up to 9 years. Based on observed structural changes in association with age, the following four-stage classification of retinal morphological changes was proposed: (I) preserved inner segment ellipsoid band (Ise), (II) disrupted ISe, (III) ISe loss and (IV) ISe and RPE loss. Data from six previously published studies reporting OCT morphology in CNGA3 and CNGB3 patients were additionally collected, forming the largest CNGA3/CNGB3 cohort to date, comprising 126 patients aged 1–71 years. Multiple regression analysis showed a significant correlation of OCT stage with age (p < 0.001) and no correlation with gene (p > 0.05). The median ages of patients with stages I–IV were 12 years, 23 years, 27 years and 48 years, respectively, and no patient older than 50 years had continuous ISe. Our findings suggest that achromatopsia presents with slowly but steadily progressive structural changes of the macular outer retinal layers. However, whether morphological changes in time follow the proposed four-stage linear pattern needs to be confirmed in a long-term study.     

Human Rod Monochromacy: Linkage Analysis and Mapping of a Cone Photoreceptor Expressed Candidate Gene on Chromosome 2q11

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at θ = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of ≈3 cM covering theACHM2locus for rod monochromacy. Radiation hybrid mapping of theCNGA3gene encoding the α-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR_10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen–D2S2222–D2S2175–(D2S2187/D2S2311)–qtel ofmarkers on 2q11 and showed that theCNGA3gene maps most closely to D2S2187 and D2S2311. These data indicate that theCNGA3gene maps within the critical interval of theACHM2locus for rod monochromacy and thus is a candidate gene for this disease.     

AAV-mediated cone rescue in a naturally occurring mouse model of CNGA3-achromatopsia

Achromatopsia is a rare autosomal recessive disorder which shows color blindness, severely impaired visual acuity, and extreme sensitivity to bright light. Mutations in the alpha subunits of the cone cyclic nucleotide-gated channels (CNGA3) are responsible for about 1/4 of achromatopsia in the U.S. and Europe. Here, we test whether gene replacement therapy using an AAV5 vector could restore cone-mediated function and arrest cone degeneration in the cpfl5 mouse, a naturally occurring mouse model of achromatopsia with a CNGA3 mutation. We show that gene therapy leads to significant rescue of cone-mediated ERGs, normal visual acuities and contrast sensitivities. Normal expression and outer segment localization of both M- and S-opsins were maintained in treated retinas. The therapeutic effect of treatment lasted for at least 5 months post-injection. This study is the first demonstration of substantial, relatively long-term restoration of cone-mediated light responsiveness and visual behavior in a naturally occurring mouse model of CNGA3 achromatopsia. The results provide the foundation for development of an AAV5-based gene therapy trial for human CNGA3 achromatopsia.     

Gene replacement therapy for retinal CNG channelopathies

Visual phototransduction relies on the function of cyclic nucleotide-gated channels in the rod and cone photoreceptor outer segment plasma membranes. The role of these ion channels is to translate light-triggered changes in the second messenger cyclic guanosine 3′–5′-monophosphate levels into an electrical signal that is further processed within the retinal network and then sent to higher visual centers. Rod and cone photoreceptors express distinct CNG channels. The rod photoreceptor CNG channel is composed of one CNGB1 and three CNGA1 subunits, whereas the cone channel is formed by one CNGB3 and three CNGA3 subunits. Mutations in any of these channel subunits result in severe and currently untreatable retinal degenerative diseases like retinitis pigmentosa or achromatopsia. In this review, we provide an overview of the human diseases and relevant animal models of CNG channelopathies. Furthermore, we summarize recent results from preclinical gene therapy studies using adeno-associated viral vectors and discuss the efficacy and translational potential of these gene therapeutic approaches.     

Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides

Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ∼2-fold greater shift in K_1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIP_n) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIP_n sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIP_n regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIP_n sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIP_n. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides.     

An ancient autosomal haplotype bearing a rare achromatopsia-causing founder mutation is shared among Arab Muslims and Oriental Jews

Numerous cultural aspects, mainly based on historical records, suggest a common origin of the Middle-Eastern Arab Muslim and Jewish populations. This is supported, to some extent, by Y-chromosome haplogroup analysis of Middle-Eastern and European samples. Up to date, no genomic regions that are shared among Arab Muslim and Jewish chromosomes and are unique to these populations have been reported. Here, we report of a rare achromatopsia-causing CNGA3 mutation (c.1585G>A) presents in both Arab Muslim and Oriental Jewish patients. A haplotype analysis of c.1585G>A-bearing chromosomes from Middle Eastern and European origins revealed a shared Muslim–Jewish haplotype, which is different from those detected in European patients, indicating a recurrent mutation stratified by a Jewish–Muslim founder effect. Comprehensive whole-genome haplotype analysis using 250 K single nucleotide polymorphism arrays revealed a large homozygous region of ~11 Mbp shared by both Arab Muslim and Oriental Jewish chromosomes. A subsequent microsatellite analysis of a 21.5 cM interval including CNGA3 and the adjacent chromosome 2 centromere revealed a unique and extremely rare haplotype associated with the c.1585G>A mutation. The age of the shared c.1585G>A mutation was calculated using the microsatellite genotyping data to be about 200 generations ago. A similar analysis of mutation age based on the Arab Muslim data alone showed that the mutation was unlikely to be the product of a recent gene flow event. The data present here demonstrate a large (11 Mbp) genomic region that is likely to originate from an ancient common ancestor of Middle-Eastern Arab Muslims and Jews who lived approximately 5,000 years ago.     

Functionally important calmodulin-binding sites in both NH2- and COOH-terminal regions of the cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit

Whereas an important aspect of sensory adaptation in rod photoreceptors and olfactory receptor neurons is thought to be the regulation of cyclic nucleotide-gated (CNG) channel activity by calcium-calmodulin (Ca2+-CaM), it is not clear that cone photoreceptor CNG channels are similarly modulated. Cone CNG channels are composed of at least two different subunit types, CNGA3 and CNGB3. We have investigated whether calmodulin modulates the activity of these channels by direct binding to the CNGB3 subunit. Heteromeric channels were formed by co-expression of human CNGB3 with human CNGA3 subunits in Xenopus oocytes; CNGB3 subunits conferred sensitivity to regulation by Ca2+-CaM, whereas CaM regulation of homomeric CNGA3 channels was not detected. To explore the mechanism underlying this regulation, we localized potential CaM-binding sites in both NH2- and COOH-terminal cytoplasmic domains of CNGB3 using gel-overlay and glutathione S-transferase pull-down assays. For both sites, binding of CaM depended on the presence of Ca2+. Individual deletions of either CaM-binding site in CNGB3 generated channels that remained sensitive to regulation by Ca2+-CaM, but deletion of both together resulted in heteromeric channels that were not modulated. Thus, both NH2- and COOH-terminal CaM-binding sites in CNGB3 are functionally important for regulation of recombinant cone CNG channels. These studies suggest a potential role for direct binding and unbinding of Ca2+-CaM to human CNGB3 during cone photoreceptor adaptation and recovery.     

CNGA3 mutations in hereditary cone photoreceptor disorders

We recently showed that mutations in the CNGA3 gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.     

Loss of CNGB1 protein leads to olfactory dysfunction and subciliary cyclic nucleotide-gated channel trapping

Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.     

Stoichiometry and assembly of olfactory cyclic nucleotide-gated channels

Native ion channels are precisely tuned to their physiological role in neuronal signaling. This tuning frequently involves the controlled assembly of heteromeric channels comprising multiple types of subunits. Cyclic nucleotide-gated (CNG) channels of olfactory neurons are tetramers and require three types of subunits, CNGA2, CNGA4, and CNGB1b, to exhibit properties necessary for olfactory transduction. Using fluorescently tagged subunits and fluorescence resonance energy transfer (FRET), we find the subunit composition of heteromeric olfactory channels in the surface membrane is fixed, with 2:1:1 CNGA2:CNGA4:CNGB1b. Furthermore, when expressed individually with CNGA2, CNGA4 and CNGB1b subunits were still present in only a single copy and, when expressed alone, did not self-assemble. These results suggest that the precise assembly of heteromeric olfactory channels results from a mechanism where CNGA4 and CNGB1b subunits have a high affinity for CNGA2 but not for self-assembly, precluding more than one CNGA4 or CNGB1b subunit in the channel complex.