Activation Of Cyclic Nucleotide-gated Channels Research Articles

T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and − 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs.9rDzNFNpqNfiW7CgYpAk_tVideo

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.

Structural and functional characterization of an achromatopsia-associated mutation in a phototransduction channel

Numerous missense mutations in cyclic nucleotide-gated (CNG) channels cause achromatopsia and retinitis pigmentosa, but the underlying pathogenic mechanisms are often unclear. We investigated the structural basis and molecular/cellular effects of R410W, an achromatopsia-associated, presumed loss-of-function mutation in human CNGA3. Cryo-EM structures of the Caenorhabditis elegans TAX-4 CNG channel carrying the analogous mutation, R421W, show that most apo channels are open. R421, located in the gating ring, interacts with the S4 segment in the closed state. R421W disrupts this interaction, destabilizes the closed state, and stabilizes the open state. CNGA3_R410W/CNGB3 and TAX4_R421W channels are spontaneously active without cGMP and induce cell death, suggesting cone degeneration triggered by spontaneous CNG channel activity as a possible cause of achromatopsia. Our study sheds new light on CNG channel allosteric gating, provides an impetus for a reevaluation of reported loss-of-function CNG channel missense disease mutations, and has implications for mutation-specific treatment of retinopathy.

CNG channel structure, function, and gating: a tale of conformational flexibility.

Cyclic nucleotide-gated (CNG) channels are key to the signal transduction machinery of certain sensory modalities both in vertebrate and invertebrate organisms. They translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through sensory cells. Despite CNG and voltage-gated potassium channels sharing a remarkable amino acid sequence homology and basic architectural plan, their functional properties are dramatically different. While voltage-gated potassium channels are highly selective and require membrane depolarization to open, CNG channels have low ion selectivity and are not very sensitive to voltage. In the last few years, many high-resolution structures of intact CNG channels have been released. This wealth of new structural information has provided enormous progress toward the understanding of the molecular mechanisms and driving forces underpinning CNG channel activation. In this review, we report on the current understanding and controversies surrounding the gating mechanism in CNG channels, as well as the deep intertwining existing between gating, the ion permeation process, and its modulation by membrane voltage. While the existence of this powerful coupling was recognized many decades ago, its direct structural demonstration, and ties to the CNG channel inherent pore flexibility, is a recent achievement.

Development of a High-Throughput Ca2+ Flux Screening Assay to Monitor Cyclic Nucleotide-Gated Channel Activity and Evaluate Achromatopsia Disease Mutant Channel Function

Development of a High-Throughput Ca2+ Flux Screening Assay to Monitor Cyclic Nucleotide-Gated Channel Activity and Evaluate Achromatopsia Disease Mutant Channel Function

Hydrophobic alkyl chains substituted to the 8-position of cyclic nucleotides enhance activation of CNG and HCN channels by an intricate enthalpy - entropy compensation

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency. The compounds were tested in homotetrameric CNGA2, heterotetrameric CNGA2:CNGA4:CNGB1b and homotetrameric HCN2 channels. We show that nearly all compounds are full agonists and that longer alkyl chains systematically increase the apparent affinity, at the best more than 30 times. The effects are stronger in CNG than HCN2 channels which, however, are constitutively more sensitive to cAMP. Kinetic analyses reveal that the off-rate is significantly slowed by the hydrophobic alkyl chains. Molecular dynamics simulations and free energy calculations suggest that an intricate enthalpy - entropy compensation underlies the higher apparent affinity of the derivatives with the longer alkyl chains, which is shown to result from a reduced loss of configurational entropy upon binding.

Biomimetic Chemical Sensing by Fluorescence Signals Using a Virus-like Particle-Based Platform.

The chemical receptors present in living organisms are promising tools for developing biomimetic chemical sensors. However, these receptors require lipid membranes for functioning under physiological conditions, which prevents their utilization in the production of cell-free in vitro chemical sensing systems. Here, we report the development of a cell-free biomimetic sensing platform using virus-like particles (VLPs) with intact ligand-gated Ca2+ channels and genetically encoded Ca2+ indicator (GECI). We observed that targeting GECI to the plasma membrane was essential for efficient loading GECI in the VLPs. Although the physiological Ca2+ concentration [Ca2+] maintained in the cells was low (∼10 nM), the concentration in the VLPs was high. This prevented the detection of the increase in [Ca2+] caused by binding of the ligand to the receptor. To address this problem, we employed Lyn-R-CEPIA1, which had low affinity for Ca2+, and a membrane targeting sequence. Thus, we succeeded in monitoring the activation of cyclic nucleotide-gated channels (CNG) on the VLPs by measuring the increase in fluorescence of Lyn-R-CEPIA1. Our VLP-based sensing system can act as a fundamental platform for all kinds of ligand-gated channels.

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.

Spectroscopic Studies of a Bacterial Cyclic Nucleotide-Gated Ion Channel

Cyclic nucleotide-gated (CNG) channels are members of the Kv channel superfamily and play a crucial role in the phototransduction and olfactory transduction pathways, where they generate the initial electrical signal following sensory transduction. CNG channels are directly regulated by cyclic nucleotides, which bind to a C-terminal cyclic nucleotide-binding domain (CNBD) connected to the pore by an intervening C-linker domain. Mutagenesis and crosslinking experiments indicate that the C-terminal region (C-linker/CNBD) undergoes a conformation change to transduce cyclic nucleotide binding into channel activation, though the exact nature of this change remains unclear. To address this question, we first sought a stable and well-behaved bacterial CNG channel suitable for spectroscopic studies. We used fluorescence-detection size-exclusion chromatography to screen a library of bacterial orthologs, which identified several candidates that could be expressed and purified at high levels. Flux studies of liposome-reconstituted channels demonstrated that these orthologs are fully functional and are activated by cyclic nucleotides. We then mutated the endogenous Cys residues in these channels before introducing Cys residues throughout the C-terminal region for spin label and fluorophore attachment. Inter-subunit double electron-electron resonance (DEER) measurements will be used to detect large-scale structural changes in these channels induced by cyclic nucleotide binding, while transition metal FRET (tmFRET) will be used to characterize subtle, localized changes to channel structure following activation. Our ultimate goal is to use these two spectroscopic techniques to characterize small- and large-scale rearrangements induced by cyclic nucleotide binding, allowing us to generate a complete structural model for CNG channel activation.

Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.

Genomic characterization, phylogenetic comparison and differential expression of the cyclic nucleotide-gated channels gene family in pear (Pyrus bretchneideri Rehd.)

The cyclic nucleotide-gated channel (CNGC) family is involved in the uptake of various cations, such as Ca2+, to regulate plant growth and respond to biotic and abiotic stresses. However, there is far less information about this family in woody plants such as pear. Here, we provided a genome-wide identification and analysis of the CNGC gene family in pear. Phylogenetic analysis showed that the 21 pear CNGC genes could be divided into five groups (I, II, III, IVA and IVB). The majority of gene duplications in pear appeared to have been caused by segmental duplication and occurred 32.94–39.14million years ago. Evolutionary analysis showed that positive selection had driven the evolution of pear CNGCs. Motif analyses showed that Group I CNGCs generally contained 26 motifs, which was the greatest number of motifs in all CNGC groups. Among these, eight motifs were shared by each group, suggesting that these domains play a conservative role in CNGC activity. Tissue-specific expression analysis indicated that functional diversification of the duplicated CNGC genes was a major feature of long-term evolution. Our results also suggested that the P-S6 and PBC & hinge domains had co-evolved during the evolution. These results provide valuable information to increase our understanding of the function, evolution and expression analyses of the CNGC gene family in higher plants.

CGMP Accumulation Causes Photoreceptor Degeneration in CNG Channel Deficiency: Evidence of cGMP Cytotoxicity Independently of Enhanced CNG Channel Function

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca(2+) influx in rod and cone photoreceptors. cGMP, the native ligand of the photoreceptor CNG channels, has been associated with cytotoxicity when its levels rise above normal due to defects in photoreceptor phosphodiesterase (PDE6) or regulation of retinal guanylyl cyclase (retGC). We found a massive accumulation of cGMP in CNGA3-deficient retina and investigated whether cGMP accumulation plays a role in cone degeneration in CNG channel deficiency. The time course study showed that the retinal cGMP level in Cnga3(-/-);Nrl(-/-) mice with CNGA3 deficiency on a cone-dominant background was sharply increased at postnatal day 8 (P8), peaked around P10-P15, remained high through P30-P60, and returned to near control level at P90. This elevation pattern correlated with photoreceptor apoptotic death, which peaked around P15-P20. In Cnga3(-/-);Gucy2e(-/-) mice lacking retGC1, cone density and expression levels of cone-specific proteins were significantly increased compared with Cnga3(-/-), consistent with a role of cGMP accumulation as the major contributor to cone death caused by CNG channel deficiency. The activity and expression levels of cGMP-dependent protein kinase G (PKG) were significantly increased in Cnga3(-/-);Nrl(-/-) retina compared with Nrl(-/-), suggesting an involvement of PKG regulation in cell death. Our results indicate that cGMP accumulation in photoreceptors can itself exert cytotoxic effect in cones, independently of CNG channel activity and Ca(2+) influx.

Extracting Modulation Transfer Function of the Visual System from Contrast Sensitivity Function in External Noise

To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells. Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3',5'-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes. Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium. These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.

Novel Role of KT5720 on Regulating Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Activity and Dorsal Root Ganglion Neuron Excitability

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in dorsal root ganglion (DRG) neurons, which are involved in diverse mechanisms that regulate DRG functions. Protein kinase A (PKA) is an essential kinase that plays a key role in almost all types of cells; it regulates the ion channel activity, the intracellular Ca(2+) concentration, as well as modulates cellular signals transduction. Nevertheless, the effect of PKA inhibition on the HCN channel activity in DRG neuron remains to be elucidated. Here we investigated the impact of PKA inhibition on the HCN channel activity and DRG neurons excitability. Our patch-clamp experiments both under whole-cell and single-channel conditions demonstrated that PKA inhibition with KT5720, a cell membrane permeable PKA-specific inhibitor, significantly attenuated HCN channel currents. Current clamp recording on freshly isolated DRG neurons showed KT5720 reduced overshoot amplitude and enhanced the threshold of the action potential. Moreover, our live-cell Ca(2+) imaging experiments illustrated KT5720 markedly reduced the intracellular Ca(2+) level. Collectively, this is the first report that addresses KT5720 attenuates the HCN channel activity and intracellular Ca(2+), thus reducing DRG neurons excitability. Therefore, our data strongly suggest that PKA is a potential target for curing HCN and DRG neuron relevant diseases.

Feedback regulation of cone cyclic nucleotide channels by phosphoinositides. Focus on “CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions”

<i>Feedback regulation of cone cyclic nucleotide channels by phosphoinositides</i>. Focus on “CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions”

Distinct Contributions of CNGA3 and CNGB3 Subunits to Ligand-Specific Activation of Cone CNG Channels

Cyclic nucleotide-gated (CNG) ion channels regulate the electrical activity of retinal photoreceptors, rods and cones, by sensing the light-induced changes of intracellular cGMP levels. Cone CNG channels consist of CNGA3 and modulatory CNGB3 subunits, both of which contain a cyclic nucleotide-binding domain (CNBD). CNGB3 subunits confer enhanced responses to cAMP and support several aspects of channel regulation. However, it is not fully understood how CNGB3 (and CNGA3) are specialized to contribute to ligand-specific activation of cone CNG channels. using patch-clamp recordings, we characterized several mutations located within the CNBD of CNGA3, each of which produced dramatic, ligand-specific effects on channel gating. In particular, D609M in CNGA3 reversed ligand selectivity, making cAMP a better agonist than cGMP, similar to equivalent mutations in paralogous channel subunits. These experiments suggest that mechanisms underlying ligand interaction with CNGA3 are well conserved. However, parallel mutations within the CNBD Cα-helix of CNGB3 had no effect on the ligand selectivity of heteromeric channels, consistent with the large decrement in sequence conservation in this region of CNGB3. CNGB3 appear to lack features supporting ligand discrimination. Next, we examined subunit contributions to ligand-dependent activation using CNBD “knock out” (R564E in CNGA3; R604E in CNGB3). CNGB3 R604E decreased relative cAMP efficacy, but only had a subtle effect on the cGMP activation for heteromeric channels (with wild-type or R564E CNGA3). In contrast, CNGA3 R564E caused an approximately 500-fold decrease in apparent cGMP affinity and nearly eliminated cAMP-dependent gating. Similar results were observed with analogous experiments using mutations of T565A in CNGA3 and T605A in CNGB3. Together, we propose that CNGA3 is the principal subunit mediating both ligand discrimination and ligand-dependent stabilization of the open state, while CNGB3 makes only a minor contribution to cGMP-dependent gating.

Insulin receptor regulates photoreceptor CNG channel activity.

Photoreceptor cyclic nucleotide gated (CNG) channels are critical elements in phototransduction and light adaptation. Here we report that insulin receptor (IR), an integral membrane protein, directly phosphorylates the CNGA1 subunit of CNG channels that in turn affects the function of these channels negatively. The IR phosphorylates Tyr(498) and Tyr(503) residues on CNGA1 that are situated at the membrane-cytoplasmic interface. The IR tyrosine kinase activity is essential for the inhibition of CNG channel. To maintain the channels in an off state, it is necessary not only to have a precise balance of the cGMP levels but also to have a control on the cGMP sensitivity of the CNG channels itself. In this study, we observed that the channel opens at a lower concentration of cGMP in IR(-/-) mice. These studies suggest that IR regulates the modulation of CNG channel activity in vivo.

Retinoic acid receptor and CNGA2 channel signaling are part of a regulatory feedback loop controlling axonal convergence and survival of olfactory sensory neurons

Little is known about the identities and functions of extracellular signaling molecules that work in concert with neuronal activity to regulate refinement and maintenance of the mouse olfactory sensory map. We show that expression of a dominant negative retinoic acid receptor (RAR) in olfactory sensory neurons (OSNs) increased the number of glomeruli that incorrectly contained OSN axons expressing different odorant receptors. This phenotype became apparent postnatally, coincided with increased cell death, and was preceded by increased Neuropilin-1 and reduced Kirrel-2 expressions. Kirrel-2-mediated cell adhesion influences odorant receptor-specific axonal convergence and is regulated by odorant receptor signaling via the olfactory cyclic nucleotide-gated (CNG) ion channel. Accordingly, we found that inhibited RAR function correlated with reduced CNG channel expression. Naris occlusion experiments and analysis of CNG channel-deficient mice further indicated that RAR-regulated CNG channel levels influenced the intrinsic neuronal activity required for cell survival in the absence of odor stimulation. Finally, we showed that CNG channel activity regulated expression of the retinoic acid-degrading enzyme Cyp26B1. Combined, these results identify a novel homeostatic feedback mechanism involving retinoic acid metabolism and CNG channel activity, which influences glomerular homogeneity and maintenance of precisely connected OSNs.—Öztokatli, H., Hörnberg, M., Berghard, A., Bohm, S. Retinoic acid receptor and CNGA2 channel signaling are part of a regulatory feedback loop controlling axonal convergence and survival of olfactory sensory neurons.

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100μM L: -cis-diltiazem or 100nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

The Glutamic Acid-Rich Protein Is a Gating Inhibitor of Cyclic Nucleotide-Gated Channels

The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors is a heterotetramer consisting of homologous CNGA1 and CNGB1a subunits. While CNGA1 is indispensable for channel activation, the specific role of CNGB1a in this process has remained elusive. Here, we show that the N-terminal glutamic acid-rich protein (GARP) domain of CNGB1a and soluble GARP2, which corresponds to the proximal portion of the GARP domain, act as autoinhibitory domains that decrease the opening probability of the CNG channel. In the presence of mutations that structurally impair the cyclic nucleotide-binding domain (CNBD) of CNGB1a, the GARP domain completely abolishes channel activity. In agreement with an inhibitory function of GARP, the activity of mutant CNG channels could be fully restored by deletion of the GARP domain. We identified two sequences within the GARP domain that confer most of the inhibitory effect and demonstrate that the profound inhibition imposed by the GARP domain is caused by direct and autonomous protein-protein interaction with the CNG channel complex. In wild-type rod CNG channels, this inhibitory effect can be relieved by binding of cGMP to the CNBD of CNGB1a. In conclusion, we propose that the N terminus of CNGB1a and soluble GARPs act as molecular gate keepers that control the activation of heteromeric rod CNG channels. Our results suggest that the GARP domain has evolved in rod photoreceptors to reduce current noise resulting from openings of CNG channels in the absence of cGMP.