Rod Cyclic Nucleotide-gated Channels Research Articles

Similar Binding Modes of cGMP Analogues Limit Selectivity in Modulating Retinal CNG Channels via the Cyclic Nucleotide-Binding Domain.

In treating retinitis pigmentosa, a genetic disorder causing progressive vision loss, selective inhibition of rod cyclic nucleotide-gated (CNG) channels holds promise. Blocking the increased Ca2+-influx in rod photoreceptors through CNG channels can potentially delay disease progression and improve the quality of life for patients. To find inhibitors for rod CNG channels, we investigated the impact of 16 cGMP analogues on both rod and cone CNG channels using the patch-clamp technique. Although modifications at the C8 position of the guanine ring did not change the ligand efficacy, modifications at the N1 and N2 positions rendered cGMP largely ineffective in activating retinal CNG channels. Notably, PET-cGMP displayed selective potential, favoring rod over cone, whereas Rp-cGMPS showed greater efficiency in activating cone over rod CNG channels. Ligand docking and molecular dynamics simulations on cyclic nucleotide-binding domains showed comparable binding energies and binding modes for cGMP and its analogues in both rod and cone CNG channels (CNGA1 vs CNGA3 subunits). Computational experiments on CNGB1a vs CNGB3 subunits showed similar binding modes albeit with fewer amino acid interactions with cGMP due to an inactivated conformation of their C-helix. In addition, no clear correlation could be observed between the computational scores and the CNG channel efficacy values, suggesting additional factors beyond binding strength determining ligand selectivity and potency. This study highlights the importance of looking beyond the cyclic nucleotide-binding domain and toward the gating mechanism when searching for selective modulators. Future efforts in developing selective modulators for CNG channels should prioritize targeting alternative channel domains.

Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel

Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM invitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.

Using cGMP analogues to modulate photoreceptor light sensitivity.

For many hereditary degenerative diseases, like retinitis pigmentosa, the primary degeneration of rod photoreceptors leads to a secondary loss of cones and eventually to blindness. The pathophysiological mechanism is very often characterized by an increase of cGMP level in rods. Thus, rod degeneration is mainly triggered by an over-activation of cyclic nucleotide-gated (CNG) channels and excessive Ca2+ influx. While targeting of CNG channels was already proposed for therapeutic purposes, thus far, it has not been possible to selectively inhibit rod CNG channels without compromising cone function. Here, we present a novel strategy, based on a combination of cGMP analogues, which enables the selective modulation of rod photoreceptor activity. This approach was first tested by means of the patch-clamp technique on retinal CNG channels, heterologously expressed in Xenopus oocytes and further confirmed by micro-electroretinography recordings on wild-type mouse retina. The cGMP-analogues mixture contained a CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and a CNG-channel activator (8-pCPT-cGMP). While Rp-8-Br-PET-cGMPS, when applied alone, behaved as a partial agonist and inhibited both cone and rod CNG channels equally, 8-pCPT-cGMP behaved as a very potent agonist and showed a strong concentration-dependent cone selectivity. Co-application of both compounds inhibited rod CNG channels and preserved cone functionality under both physiological and pathological cGMP levels. While the kinetics of rod channels was not influenced by this treatment, the deactivation of cone channels was slowed down. Surprisingly, this aspect did not affect the physiological cone responsiveness to light, indicating possible intracellular compensatory mechanisms. In conclusion, by delaying primary rods degeneration, this strategy shields the healthy cones and thus may either postpone the onset or slow down the disease development. Beyond the treatment of retinal diseases, using cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies of related diseases.

Biology, Pathobiology and Gene Therapy of CNG Channel-Related Retinopathies

The visual process begins with the absorption of photons by photopigments of cone and rod photoreceptors in the retina. In this process, the signal is first amplified by a cyclic guanosine monophosphate (cGMP)-based signaling cascade and then converted into an electrical signal by cyclic nucleotide-gated (CNG) channels. CNG channels are purely ligand-gated channels whose activity can be controlled by cGMP, which induces a depolarizing Na+/Ca2+ current upon binding to the channel. Structurally, CNG channels belong to the superfamily of pore-loop cation channels and share structural similarities with hyperpolarization-activated cyclic nucleotide (HCN) and voltage-gated potassium (KCN) channels. Cone and rod photoreceptors express distinct CNG channels encoded by homologous genes. Mutations in the genes encoding the rod CNG channel (CNGA1 and CNGB1) result in retinitis-pigmentosa-type blindness. Mutations in the genes encoding the cone CNG channel (CNGA3 and CNGB3) lead to achromatopsia. Here, we review the molecular properties of CNG channels and describe their physiological and pathophysiological roles in the retina. Moreover, we summarize recent activities in the field of gene therapy aimed at developing the first gene therapies for CNG channelopathies.

Retinal Cyclic Nucleotide-Gated Channel Regulation by Calmodulin.

Retinal cyclic nucleotide-gated (CNG) ion channels bind to intracellular cGMP and mediate visual phototransduction in photoreceptor rod and cone cells. Retinal rod CNG channels form hetero-tetramers comprised of three CNGA1 and one CNGB1 protein subunits. Cone CNG channels are similar tetramers consisting of three CNGA3 and one CNGB3 subunits. Calmodulin (CaM) binds to two distinct sites (CaM1: residues 565-587 and CaM2: residues 1120-1147) within the cytosolic domains of rod CNGB1. The binding of Ca2+-bound CaM to CNGB1 promotes the Ca2+-induced desensitization of CNG channels in retinal rods that may be important for photoreceptor light adaptation. Mutations that affect Ca2+-dependent CNG channel function are responsible for inherited forms of blindness. In this review, we propose structural models of the rod CNG channel bound to CaM that suggest how CaM might cause channel desensitization and how dysregulation of the channel may lead to retinal disease.

CGMP Analogues with Opposing Actions on CNG Channels Selectively Modulate Rod or Cone Photoreceptor Function.

The vertebrate retina harbors rod and cone photoreceptors. Human vision critically depends on cone photoreceptor function. In the phototransduction cascade, cGMP activates distinct rod and cone isoforms of the cyclic nucleotide-gated (CNG) channel. Excessive cGMP levels initiate a pathophysiological rollercoaster, which starts with CNG channel over-activation, typically in rod photoreceptors. This triggers cell death of rods first, and then cones, and is the root cause of many blinding retinal diseases, including Retinitis pigmentosa. While targeting of CNG channels has been proposed for therapeutic purposes, thus far, it has not been possible to inhibit rod CNG channels without compromising cone function. Here, we present a novel strategy, based on cGMP analogues with opposing actions on CNG channels, which enables the selective modulation of either rod or cone photoreceptor activity. The combined treatment with the weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and the cone-selective CNG-channel activator (8-pCPT-cGMP) essentially normalized rod CNG-channel function while preserving cone functionality at physiological and pathological cGMP levels. Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Moreover, this strategy may allow for improvements in visual performance in certain light environments.

The Natural History of CNGB1-Related Retinopathy: A Longitudinal Phenotypic Analysis.

Cyclic nucleotide-gated channel β 1 (CNGB1) encodes a subunit of the rod cyclic nucleotide-gated channel. Pathogenic variants in CNGB1 are responsible for 4% of autosomal recessive retinitis pigmentosa (RP). Several treatment strategies show promise for treating inherited retinal degenerations, however relevant metrics of progression and sensitive clinical trial endpoints are needed to assess therapeutic efficacy. This study reports the natural history of CNGB1-related RP with a longitudinal phenotypic analysis of 33 molecularly-confirmed patients with a mean follow-up period of 4.5 ± 3.9 years (range 0–17). The mean best corrected visual acuity (BCVA) of the right eye was 0.31 ± 0.43 logMAR at baseline and 0.47 ± 0.63 logMAR at the final visit over the study period. The ellipsoid zone (EZ) length was measurable in at least one eye of 23 patients and had a mean rate of constriction of 178 ± 161 µm per year (range 1.0–661 µm), with 57% of patients having a decrease in EZ length of greater than 250 µm in a simulated two-year trial period. Hyperautofluorescent outer ring (hyperAF) area was measurable in 17 patients, with 10 patients not displaying a ring phenotype. The results support previous findings of CNGB1-related RP being a slowly progressive disease with patients maintaining visual acuity. Prospective deep phenotyping studies assessing multimodal retinal imaging and functional measures are now required to determine clinical endpoints to be used in a trial.

Inherited Retinal Degeneration: PARP-Dependent Activation of Calpain Requires CNG Channel Activity.

Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b-mutant rd1 mouse and rd1*Cngb1−/− double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1*Cngb1−/− double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b-dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases.

Chemical shift assignments of calmodulin bound to the β-subunit of a retinal cyclic nucleotide-gated channel (CNGB1)

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679–702) binds tightly to Ca2+-bound CaM, which promotes Ca2+-induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222).

The structure of the native CNGA1/CNGB1 CNG channel from bovine retinal rods.

In rod photoreceptors of the retina, the cyclic nucleotide-gated (CNG) channel is composed of three CNGA and one CNGB subunits, and it closes in response to light activation to generate an electrical signal that is conveyed to the brain. Here we report the cryo-EM structure of the closed state of the native rod CNG channel isolated from bovine retina. The structure reveals differences between CNGA1 and CNGB1 subunits. Three CNGA1 subunits are tethered at their C terminus by a coiled-coil region. The C-helix in the cyclic nucleotide-binding domain of CNGB1 features a different orientation from that in the three CNGA1 subunits. The arginine residue R994 of CNGB1 reaches into the ionic pathway and blocks the pore, thus introducing an additional gate, which is different from the central hydrophobic gate known from homomeric CNGA channels. These results address the long-standing question of how CNGB1 subunits contribute to the function of CNG channels in visual and olfactory neurons.

Structural mechanisms of assembly, permeation, gating, and pharmacology of native human rod CNG channel

Mammalian cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cGMP or cAMP and play essential roles in the signal transduction of the visual and olfactory sensory systems. CNGA1, the principal component of the CNG channel from rod photoreceptors, can by itself form a functional homotetrameric channel and has been used as the model system in the majority of rod CNG studies. However, the native rod CNG functions as a heterotetramer consisting of three A1 and one B1 subunits and exhibits different functional properties than the CNGA1 homomer. Here we present the functional analysis of human rod CNGA1/B1 heterotetramer and its cryo-EM structures in apo, cGMP-bound, cAMP-bound, and L-cis-Diltiazem-blocked states. These structures, with resolution ranging from 2.6 to 3.3Å, elucidate the structural mechanisms underlying the 3:1 subunit stoichiometry, the asymmetrical gating upon cGMP activation, and the unique pharmacological property of the native rod CNG channel.

The GARP Domain of the Rod CNG Channel's β1-Subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disk Rim.

Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disk-shaped membranes. Disk membranes are separated from the surrounding plasma membrane, and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane, where it mediates the electrical response to light. Using Xenopus and mouse models of both sexes, we now show that the targeted delivery of the CNG channel to the outer segment uses the conventional secretory pathway, including protein processing in both ER and Golgi, and requires preassembly of its constituent α1 and β1 subunits. We further demonstrate that the N-terminal glutamic acid-rich protein (GARP) domain of CNGβ1 contains two distinct functional regions. The glutamic acid-rich region encodes specific information targeting the channel to rod outer segments. The adjacent proline-enriched region connects the CNG channel to photoreceptor disk rims, likely through an interaction with peripherin-2. These data reveal fine functional specializations within the structural domains of the CNG channel and suggest that its sequestration to the outer segment plasma membrane requires an interaction with peripherin-2.SIGNIFICANCE STATEMENT Neurons and other differentiated cells have a remarkable ability to deliver and organize signaling proteins at precise subcellular locations. We now report that the CNG channel, mediating the electrical response to light in rod photoreceptors, contains two specialized regions within the N terminus of its β-subunit: one responsible for delivery of this channel to the ciliary outer segment organelle and another for subsequent channel sequestration into the outer segment plasma membrane. These findings expand our understanding of the molecular specializations used by neurons to populate their critical functional compartments.

Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice.

Retinitis pigmentosa is an inherited blinding disorder characterized by progressive degeneration and loss of photoreceptors. The exact mechanism of degeneration and cell death of photoreceptors is not known, but is thought to involve disturbed Ca2+—signaling. Ca2+ can enter the photoreceptor cell via outer segment cyclic nucleotide-gated (CNG) channels or synaptic Cav1.4 L-type voltage-gated calcium channels (VGCC). Previously, we have shown that genetic ablation of the Cngb1 gene encoding the B subunit of the rod CNG channel delays the fast progressing degeneration in the rd1 mutant mouse model of retinitis pigmentosa. In this study, we crossbred rd1 mice with the Cacna1f-deficient mouse lacking the Cav1.4 α1 subunit of the L-type VGCC. Longitudinal in vivo examinations of photoreceptor layer thickness by optical coherence tomography revealed a significant, but not sustained delay of retinal degeneration in Cacna1f x rd1 double mutant mice compared to rd1 mice. This was accompanied by a reduction of TUNEL positive cells in the early phase of rod degeneration. Remarkably, Cacna1f x rd1 double mutant mice displayed a strong decrease in the activation of the Ca2+-dependent protease calpain during photoreceptor loss. Our results show that genetic deletion of the synaptic Cav1.4 L-type VGCCs impairs calpain activation and leads to a short-term preservation of photoreceptors in the rd1 mouse.

Numb regulates the polarized delivery of cyclic nucleotide-gated ion channels in rod photoreceptor cilia.

The development and maintenance of protein compartmentalization is essential for neuronal function. A striking example is observed in light-sensing photoreceptors, in which the apical sensory cilium is subdivided into an inner and outer segment, each containing specific proteins essential for vision. It remains unclear, however, how such polarized protein localization is regulated. We report here that the endocytic adaptor protein Numb localizes to the inner, but not the outer segment of mouse photoreceptor cilia. Rod photoreceptor-specific inactivation of numb in vivo leads to progressive photoreceptor degeneration, indicating an essential role for Numb in photoreceptor cell biology. Interestingly, we report that loss of Numb in photoreceptors does not affect the localization of outer segment disk membrane proteins, such as rhodopsin, Peripherin-rds, Rom-1, and Abca4, but significantly disrupts the localization of the rod cyclic nucleotide-gated (Cng) channels, which accumulates on the inner segment plasma membrane in addition to its normal localization to the outer segments. Mechanistically, we show that Numb interacts with both subunits of the Cng channel and promotes the trafficking of Cnga1 to the recycling endosome. These results suggest a model in which Numb prevents targeting of Cng channels to the inner segment, by promoting their trafficking through the recycling endosome, where they can be sorted for specific delivery to the outer segment. This study uncovers a novel mechanism regulating polarized protein delivery in light-sensing cilia, raising the possibility that Numb plays a part in the regulation of protein trafficking in other types of cilia.

Peripherin-2 couples rhodopsin to the CNG channel in outer segments of rod photoreceptors

Outer segments (OSs) of rod photoreceptors are cellular compartments specialized in the conversion of light into electrical signals. This process relies on the light-triggered change in the intracellular levels of cyclic guanosine monophosphate, which in turn controls the activity of cyclic nucleotide-gated (CNG) channels in the rod OS plasma membrane. The rod CNG channel is a macromolecular complex that in its core harbors the ion-conducting CNGA1 and CNGB1a subunits. To identify additional proteins of the complex that interact with the CNGB1a core subunit, we applied affinity purification of mouse retinal proteins followed by mass spectrometry. In combination with in vitro and in vivo co-immunoprecipitation and fluorescence resonance energy transfer (FRET), we found that the tetraspanin peripherin-2 links CNGB1a to the light-detector rhodopsin. Using immunoelectron microscopy, we found that this peripherin-2/rhodopsin/CNG channel complex localizes to the contact region between the disk rims and the plasma membrane. FRET measurements revealed that the fourth transmembrane domain (TM4) of peripherin-2 is required for the interaction with rhodopsin. Quantitatively, the binding affinity of the peripherin-2/rhodopsin interaction was in a similar range as that observed for rhodopsin dimers. Finally, we demonstrate that the p.G266D retinitis pigmentosa mutation found within TM4 selectively abolishes the binding of peripherin-2 to rhodopsin. This finding suggests that the specific disruption of the rhodopsin/peripherin-2 interaction in the p.G266D mutant might contribute to the pathophysiology in affected persons.

Submembrane assembly and renewal of rod photoreceptor cGMP-gated channel: insight into the actin-dependent process of outer segment morphogenesis.

The photoreceptor outer segment (OS) is comprised of two compartments: plasma membrane (PM) and disk membranes. It is unknown how the PM renewal is coordinated with that of the disk membranes. Here we visualized the localization and trafficking process of rod cyclic nucleotide-gated channel α-subunit (CNGA1), a PM component essential for phototransduction. The localization was visualized by fusing CNGA1 to a fluorescent protein Dendra2 and expressing in Xenopus laevis rod photoreceptors. Dendra2 allowed us to label CNGA1 in a spatiotemporal manner and therefore discriminate between old and newly trafficked CNGA1-Dendra2 in the OS PM. Newly synthesized CNGA1 was preferentially trafficked to the basal region of the lateral OS PM where newly formed and matured disks are also added. Unique trafficking pattern and diffusion barrier excluded CNGA1 from the PM domains, which are the proposed site of disk membrane maturation. Such distinct compartmentalization allows the confinement of cyclic nucleotide-gated channel in the PM, while preventing the disk membrane incorporation. Cytochalasin D and latrunculin A treatments, which are known to disrupt F-actin-dependent disk membrane morphogenesis, prevented the entrance of newly synthesized CNGA1 to the OS PM, but did not prevent the entrance of rhodopsin and peripherin/rds to the membrane evaginations believed to be disk membrane precursors. Uptake of rhodopsin and peripherin/rds coincided with the overgrowth of the evaginations at the base of the OS. Thus F-actin is essential for the trafficking of CNGA1 to the ciliary PM, and coordinates the formations of disk membrane rim region and OS PM.

Cyclic Nucleotide-Gated Channel Subunit Glycosylation Regulates Matrix Metalloproteinase-Dependent Changes in Channel Gating

Cyclic-nucleotide gated (CNG) channels are essential for phototransduction within retinal photoreceptors. We have demonstrated previously that the enzymatic activity of matrix metalloproteinase-2 and -9, members of the matrix metalloproteinase (MMP) family of extracellular, Ca(2+)- and Zn(2+)-dependent proteases, enhances the ligand sensitivity of both rod (CNGA1 and CNGB1) and cone (CNGA3 and CNGB3) CNG channels. Additionally, we have observed a decrease in the maximal CNG channel current (Imax) that begins late during MMP-directed gating changes. Here we demonstrate that CNG channels become nonconductive after prolonged MMP exposure. Concurrent with the loss of conductive channels is the increased relative contribution of channels exhibiting nonmodified gating properties, suggesting the presence of a subpopulation of channels that are protected from MMP-induced gating effects. CNGA subunits are known to possess one extracellular core glycosylation site, located at one of two possible positions within the turret loop near the pore-forming region. Our results indicate that CNGA glycosylation can impede MMP-dependent modification of CNG channels. Furthermore, the relative position of the glycosylation site within the pore turret influences the extent of MMP-dependent proteolysis. Glycosylation at the site found in CNGA3 subunits was found to be protective, while glycosylation at the bovine CNGA1 site was not. Relocating the glycosylation site in CNGA1 to the position found in CNGA3 recapitulated CNGA3-like protection from MMP-dependent processing. Taken together, these data indicate that CNGA glycosylation may protect CNG channels from MMP-dependent proteolysis, consistent with MMP modification of channel function having a requirement for physical access to the extracellular face of the channel.

Gene therapy restores vision and delays degeneration in the CNGB1-/- mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.

Matrix Metalloproteinase-9 Increases the Ligand Sensitivity of CNG Channels: Evidence for Sequential Subunit Modification

Matrix metalloproteinases (MMPs) are a family of over 20 cell-surface and secreted, Zn2+- and Ca2+-dependent endopeptidases. Two closely related gelatinase MMPs, −9 and −2, have been implicated in both normal retinal processes and in several retinal diseases. MMPs are classically associated with tissue remodeling, but have been shown to influence ion channel function—either indirectly (e.g., through modification of the pericellular environment) or directly (e.g., by proteolysis of channel subunits). Because MMPs have been localized within the interphotoreceptor matrix, we hypothesized that MMP catalytic activity can influence the function of photoreceptor cyclic nucleotide-gated (CNG) channels. Cone and rod CNG channels were individually expressed in Xenopus oocytes and examined via patch-clamp recording in the inside-out configuration. Extracellular exposure to MMP-9 or −2 produced a profound increase in the efficacy of cAMP and the apparent cGMP affinity for both homomeric (CNGA3, K1/2,GMP, mean ± S.E.M: MMP960min = 0.62 μM ± 0.21; control60min = 12.6 μM ± 1.7) and heteromeric (CNGA3+CNGB3, K1/2,GMP: MMP980min = 2.2 μM ± 0.6; control80min = 16.4 μM ± 1.2) channel compositions. The gating changes required MMP-catalytic activity, exhibited complex state-dependence and were attenuated by N-glycosylation in the pore turret of CNGA subunits. Furthermore, MMP exposure resulted in decreased abundance of the full length CNGA3 subunit on immunoblots, and a concomitant increase in putative degradation products, consistent with proteolysis of core-channel subunits. Analyses of cGMP dose-response curves and current fluctuations suggest that MMPs generate channel subpopulations by sequentially modifying individual CNG channel subunits, where the apparent affinity for cGMP is governed by the total number of modified subunits (from 0 to 4). Together, these results provide evidence that MMPs can incrementally regulate the ligand sensitivity of CNG channels by proteolytic modification of CNGA subunits.