The Experts below are selected from a list of 840 Experts worldwide ranked by ideXlab platform
William N. Zagotta - One of the best experts on this subject based on the ideXlab platform.
-
regulation of cnga1 channel gating by interactions with the membrane
Journal of Biological Chemistry, 2016Co-Authors: Teresa K Aman, Sharona E Gordon, William N. ZagottaAbstract:Abstract Cyclic nucleotide-gated (CNG) channels are expressed in rod photoreceptors and open in response to direct binding of cyclic nucleotides. We have previously shown that potentiation of CNGA1 channels by transition metals requires a histidine in the A′ helix following the S6 transmembrane segment. Here, we used transition metal ion FRET and patch clamp fluorometry with a fluorescent, noncanonical amino acid (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap)) to show that the potentiating transition metal Co2+ binds in or near the A′ helix. Adding high-affinity metal-binding sites to the membrane (stearoyl-nitrilotriacetic acid (C18-NTA)) increased potentiation for low Co2+ concentrations, indicating that the membrane can coordinate metal ions with the A′ helix. These results suggest that restraining the A′ helix to the plasma membrane potentiates CNGA1 channel opening. Similar interactions between the A′ helix and the plasma membrane may underlie regulation of structurally related hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium subfamily H (KCNH) channels by plasma membrane components.
-
regulation of cnga1 channel gating by interactions with the membrane
Biophysical Journal, 2016Co-Authors: Teresa K Aman, Sharona E Gordon, William N. ZagottaAbstract:We have previously shown that a histidine (H420) in the first intracellular helix (A’ helix) of cyclic nucleotide-gated (CNGA1) channels is involved in potentiation of CNGA1 currents by transition metal ions. The binding of transition metals stabilizes the open state of the channel, so understanding the details of metal ion coordination will provide insight into the structural changes that occur after ligand binding to promote channel opening. Because structural studies suggest that the A’ helix is proximal to the membrane, we tested the hypothesis that a metal ion is coordinated between H420 and the intracellular leaflet of the bilayer with transition metal FRET (tmFRET) and patch-clamp fluorometry. To label CNGA1 channels, the amber codon suppression strategy was used to incorporate a fluorescent, noncanonical amino acid, L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap) near H420. We chose Co2+ (1-30 μM) to potentiate CNGA1 currents and serve as a FRET acceptor for L-Anap in inside-out patches from Xenopus oocytes. We found that Co2+ quenched Anap fluorescence by ∼50%, and the H420Q mutation significantly attenuated quenching, providing further evidence that Co2+ binds to H420. Next, to test whether the membrane provides a second Co2+ ligand, we added high-affinity metal binding sites to the membrane using a novel, metal-chelating fatty acid (C18-NTA). We found that metal potentiation was increased for low (1-3 μM) Co2+ concentrations with C18-NTA-Co2+, and this effect required H420. These results indicate that membrane-bound metal ions interact with the A’ helix, suggesting that the mechanism for metal potentiation is metal ion coordination between H420 and a lipid with an anionic headgroup with lower affinity for Co2+ compared to C18-NTA. This arrangement of the A’ helix relative to the membrane potentiates channel opening and may be an important structural transition that occurs with cyclic nucleotide regulation.
-
The Journal of General Physiology
2013Co-Authors: Matthew C Trudeau, William N. ZagottaAbstract:abstract Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca 2 � ions. Rod CNG channels are directly inhibited by Ca 2 �-calmodulin (Ca 2 � /CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca 2 � /CaM-dependent modulation. The molecular basis for Ca 2 � /CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca 2 � /CaM to a site in the NH 2-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH 2-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca 2 � /CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca 2 � /CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH 2- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca 2 � /CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH 2- and COOH-terminal regions underlies Ca 2 � /CaMdependent inhibition. key words
-
dynamics of ca2 calmodulin dependent inhibition of rod cyclic nucleotide gated channels measured by patch clamp fluorometry
The Journal of General Physiology, 2004Co-Authors: Matthew C Trudeau, William N. ZagottaAbstract:Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca2+ ions. Rod CNG channels are directly inhibited by Ca2+-calmodulin (Ca2+/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca2+/CaM-dependent modulation. The molecular basis for Ca2+/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca2+/CaM to a site in the NH2-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH2-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca2+/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca2+/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH2- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca2+/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH2- and COOH-terminal regions underlies Ca2+/CaM-dependent inhibition.
-
rod cyclic nucleotide gated channels have a stoichiometry of three cnga1 subunits and one CNGB1 subunit
Neuron, 2002Co-Authors: Jie Zheng, Matthew C Trudeau, William N. ZagottaAbstract:Abstract Phototransduction relies on the precise balance of speed and sensitivity to achieve optimal performance. The cyclic nucleotide-gated (CNG) ion channels, with their Ca 2+ permeability, high sensitivity to changes in cytosolic cGMP, rapid gating kinetics, and Ca 2+ -calmodulin modulation, are beautifully optimized for their role in light detection. Many of these specializations come about from the heteromeric composition of the native channel, comprised of CNGA1 and CNGB1 subunits. However, the stoichiometry and arrangement of these subunits is unknown. Here we have used an approach based on fluorescence resonance energy transfer (FRET) to determine the composition of the intact functional channel in the surface membrane. We find, surprisingly, that the channel contains three CNGA1 subunits and only one CNGB1 subunit. These results have implications for CNG channel function in particular and assembly of membrane proteins in general.
Jacqueline C. Tanaka - One of the best experts on this subject based on the ideXlab platform.
-
A Cyclic Nucleotide-Gated Channel Mutation Associated with Canine Daylight Blindness Provides Insight into a Role for the S2 Segment Tri-Asp motif in Channel
2016Co-Authors: Naoto Tanaka, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helica
-
A cyclic nucleotide-gated channel mutation associated with canine daylight blindness provides insight into a role for the S2 segment Tri-Asp motif in channel biogenesis
PloS one, 2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1–S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis.
-
Sequence alignment of the S2 domain of the cB3 subunit residues define the Tri-Asp motif.
2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Asp252, 256 and 262 in canine B3 delineate the highly conserved Tri-Asp motif which is conserved in all CNG channels with relative positions 2 and 3 conserved in the HCN channels. The flanking positions 1 and 3 are acidic residues in the Kv channels aligned here. c = canine; b = bovine; h = human, r = rat; m = mouse. CNGA1 and CNGB1 are rod channels; CNGA2 and CNGA4 are channel subunits expressed in olfactory cilia.
-
A Canine CNGB3 Channelopathy Suggests that Changes in Calcium Homeostasis Result in Progressive Loss of Cone Function
Biophysical Journal, 2013Co-Authors: Naoto Tanaka, Andras M Komaromy, Amalaris Gonzalez, Jacqueline C. TanakaAbstract:Canine day-blindness, a model for human achromatopsia, is associated with loss of cone function due either to the deletion or a missense Asp (D) 262 to Asn (N) mutation in CNGB3. Asp 262 resides in an acidic motif in the S2 transmembrane helix conserved in all CNG channel subunits and members of the Shaker K+ superfamily. Tetrameric cyclic nucleotide-gated (CNG) channels are formed from CNGA3 and CNGB3 subunits and transduce light information in cone photoreceptor outer segments. In canine day-blindness, the CNGB3-D262N mutation leads to loss of cone function between 4 and ∼10 weeks suggesting progressive physiological changes. We investigated the missense mutation using the human CNGB3, previously used in gene therapy to restore cone function in young dogs (Hum Mol Genet 2010 19: 2581). Canine CNGA3 was co-expressed with hCNGB3; the most significant functional difference between homomeric and heteromeric currents was an ∼10 fold increase in PCa/PNa in heteromeric channels. Co-expression of cCNGA3 with hCNGB3-D262N (canine numbering) result in the absence of functional heteromeric channels with evidence of some homomeric CNGA3 channels. We suggest that alterations in calcium homeostasis associated with the missense mutation in CNGB3 contribute to the loss of cone function. We generated mutations in the Asp residues in CNGA3 channels. We investigated substitutions in the three Asp residues in S2 and all mutations examined resulted in the loss of channel function underscoring the essential role for these residues in channel function. Studies in voltage-gated channels show electrostatic interactions between the acidic residues in S2 and residues in S3 and S4 transmembrane domains. Our future experiments will explore the role of these acidic residues in intra-subunit helical interactions using mutagenesis and molecular modeling.
Naoto Tanaka - One of the best experts on this subject based on the ideXlab platform.
-
A Cyclic Nucleotide-Gated Channel Mutation Associated with Canine Daylight Blindness Provides Insight into a Role for the S2 Segment Tri-Asp motif in Channel
2016Co-Authors: Naoto Tanaka, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helica
-
A cyclic nucleotide-gated channel mutation associated with canine daylight blindness provides insight into a role for the S2 segment Tri-Asp motif in channel biogenesis
PloS one, 2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1–S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis.
-
Sequence alignment of the S2 domain of the cB3 subunit residues define the Tri-Asp motif.
2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Asp252, 256 and 262 in canine B3 delineate the highly conserved Tri-Asp motif which is conserved in all CNG channels with relative positions 2 and 3 conserved in the HCN channels. The flanking positions 1 and 3 are acidic residues in the Kv channels aligned here. c = canine; b = bovine; h = human, r = rat; m = mouse. CNGA1 and CNGB1 are rod channels; CNGA2 and CNGA4 are channel subunits expressed in olfactory cilia.
-
A Canine CNGB3 Channelopathy Suggests that Changes in Calcium Homeostasis Result in Progressive Loss of Cone Function
Biophysical Journal, 2013Co-Authors: Naoto Tanaka, Andras M Komaromy, Amalaris Gonzalez, Jacqueline C. TanakaAbstract:Canine day-blindness, a model for human achromatopsia, is associated with loss of cone function due either to the deletion or a missense Asp (D) 262 to Asn (N) mutation in CNGB3. Asp 262 resides in an acidic motif in the S2 transmembrane helix conserved in all CNG channel subunits and members of the Shaker K+ superfamily. Tetrameric cyclic nucleotide-gated (CNG) channels are formed from CNGA3 and CNGB3 subunits and transduce light information in cone photoreceptor outer segments. In canine day-blindness, the CNGB3-D262N mutation leads to loss of cone function between 4 and ∼10 weeks suggesting progressive physiological changes. We investigated the missense mutation using the human CNGB3, previously used in gene therapy to restore cone function in young dogs (Hum Mol Genet 2010 19: 2581). Canine CNGA3 was co-expressed with hCNGB3; the most significant functional difference between homomeric and heteromeric currents was an ∼10 fold increase in PCa/PNa in heteromeric channels. Co-expression of cCNGA3 with hCNGB3-D262N (canine numbering) result in the absence of functional heteromeric channels with evidence of some homomeric CNGA3 channels. We suggest that alterations in calcium homeostasis associated with the missense mutation in CNGB3 contribute to the loss of cone function. We generated mutations in the Asp residues in CNGA3 channels. We investigated substitutions in the three Asp residues in S2 and all mutations examined resulted in the loss of channel function underscoring the essential role for these residues in channel function. Studies in voltage-gated channels show electrostatic interactions between the acidic residues in S2 and residues in S3 and S4 transmembrane domains. Our future experiments will explore the role of these acidic residues in intra-subunit helical interactions using mutagenesis and molecular modeling.
Stylianos Michalakis - One of the best experts on this subject based on the ideXlab platform.
-
identification of a common non apoptotic cell death mechanism in hereditary retinal degeneration
PLOS ONE, 2014Co-Authors: Blanca Arangogonzalez, Stylianos Michalakis, Susanne Koch, Ayse Sahaboglu, Katharina Kranz, Pietro Farinelli, Fred Koch, Sandra Cottet, Dragana Trifunovic, Ulrike JanssenbienholdAbstract:Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, CNGB1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.
-
Peripherin-2 couples rhodopsin to the CNG channel in outer segments of rod photoreceptors
Human molecular genetics, 2014Co-Authors: Elvir Becirovic, O. N. Phuong Nguyen, Christian Wahl-schott, Elisabeth Butz, Uwe Wolfrum, Christos Paparizos, Gabi Stern-schneider, Marius Ueffing, Stylianos MichalakisAbstract: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.
-
RD animal models used and their genetic defects.
2014Co-Authors: Blanca Arango-gonzalez, Stylianos Michalakis, Susanne Koch, Dragana Trifunović, Ayse Sahaboglu, Katharina Kranz, Pietro Farinelli, Fred Koch, Sandra Cottet, Ulrike Janssen-bienholdAbstract:The cartoon illustrates the anatomical localization and metabolic consequences of the causative genetic mutations in the ten different RD animal models used in this study. RD causing mutations in these animal models interfere with the various stages of the phototransduction cascade, from the 11-cis-retinal recycling enzyme RPE65 (Rpe65 KO), via the light-sensitive Rhodopsin (Rho KO, P23H, S334ter), cGMP-hydrolyzing phosphodiesterase-6 (PDE6; rd1, rd10, cpfl1), the structural protein Peripherin (Prph2; rd2), to the cyclic-nucleotide-gated (CNG; CNGB1 KO, Cnga3 KO) channel that allows for Ca2+-influx.
-
Gene replacement therapy for retinal CNG channelopathies
Molecular Genetics and Genomics, 2013Co-Authors: Christian Schön, Martin Biel, Stylianos MichalakisAbstract: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.
-
c.3444+1G>A mutation affects the splicing and expression of CNGB1.
2013Co-Authors: Elvir Becirovic, Martin Biel, Kostadinka Nakova, Verena Hammelmann, Roman Hennel, Stylianos MichalakisAbstract:(A) Schematic representation of the minigene construct used for the exon trapping experiment showing the position of the c.3444+1G>A mutation (marked by an arrowhead) and the deleted intronic XbaI-fragment. Vector backbone sequence is depicted in green. (B) Revese transcriptase PCR from HEK293T cells transfected with mutant and wild type minigene constructs. The electropherogram for the c.3444+1G>A mutant shows the skipping of exon 32. (C) Scheme showing the splice products. The length of the respective PCR products is indicated by double arrows. (D) Schematic comparison of the WT and mutant protein demonstrating the lack of the entire distal C-terminus and the last 10 aa of the αC helix in the context of the c.3444G>A mutation. Skipping of exon 32 causes a frameshift which results in addition of 68 unrelated amino acids after aa position 1075 of the CNGB1a protein (highlighted in grey). The numbers represent the length of the respective proteins (1245 aa for WT and 1143 for the mutant). (E) Western blot of membranes isolated from HEK293T cells transfected with CNGA1 and wild type or mutant CNGB1a probed with anti-B1 (top panel) or anti-ATPase (bottom panel). The weaker expression of the mutant protein was normalized in the presence of the proteasome inhibitors MG-132 and ALLN. CNBD: cyclic nucleotide-binding domain. Primers are shown as arrows. S1–S6: transmembrane segments; WT: wild type, Mut: c.3444+1G>A mutation.
Martin Biel - One of the best experts on this subject based on the ideXlab platform.
-
Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia
Human molecular genetics, 2016Co-Authors: Christian Schön, Sabrina Asteriti, Susanne Koch, Vithiyanjali Sothilingam, Marina Garcia Garrido, Naoyuki Tanimoto, Jochen Herms, Mathias W. Seeliger, Lorenzo Cangiano, Martin BielAbstract:Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in CNGB1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in CNGB1 KO mice. Patch-clamp recordings revealed that the membrane potentials of CNGB1 KO and CNGB1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in CNGB1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.
-
Gene replacement therapy for retinal CNG channelopathies
Molecular Genetics and Genomics, 2013Co-Authors: Christian Schön, Martin Biel, Stylianos MichalakisAbstract: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.
-
c.3444+1G>A mutation affects the splicing and expression of CNGB1.
2013Co-Authors: Elvir Becirovic, Martin Biel, Kostadinka Nakova, Verena Hammelmann, Roman Hennel, Stylianos MichalakisAbstract:(A) Schematic representation of the minigene construct used for the exon trapping experiment showing the position of the c.3444+1G>A mutation (marked by an arrowhead) and the deleted intronic XbaI-fragment. Vector backbone sequence is depicted in green. (B) Revese transcriptase PCR from HEK293T cells transfected with mutant and wild type minigene constructs. The electropherogram for the c.3444+1G>A mutant shows the skipping of exon 32. (C) Scheme showing the splice products. The length of the respective PCR products is indicated by double arrows. (D) Schematic comparison of the WT and mutant protein demonstrating the lack of the entire distal C-terminus and the last 10 aa of the αC helix in the context of the c.3444G>A mutation. Skipping of exon 32 causes a frameshift which results in addition of 68 unrelated amino acids after aa position 1075 of the CNGB1a protein (highlighted in grey). The numbers represent the length of the respective proteins (1245 aa for WT and 1143 for the mutant). (E) Western blot of membranes isolated from HEK293T cells transfected with CNGA1 and wild type or mutant CNGB1a probed with anti-B1 (top panel) or anti-ATPase (bottom panel). The weaker expression of the mutant protein was normalized in the presence of the proteasome inhibitors MG-132 and ALLN. CNBD: cyclic nucleotide-binding domain. Primers are shown as arrows. S1–S6: transmembrane segments; WT: wild type, Mut: c.3444+1G>A mutation.
-
endoplasmic reticulum stress associated cone photoreceptor degeneration in cyclic nucleotide gated channel deficiency
Journal of Biological Chemistry, 2012Co-Authors: Arjun Thapa, Stylianos Michalakis, Martin Biel, Lynsie Morris, Xiqin DingAbstract: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-IP3R, 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.
-
The Retinitis Pigmentosa Mutation c.3444+1G.A in CNGB1 Results in Skipping of Exon 32
2010Co-Authors: Elvir Becirovic, Kostadinka Nakova, Verena Hammelmann, Roman Hennel, Martin BielAbstract:Retinitis pigmentosa (RP) is a severe hereditary eye disorder characterized by progressive degeneration of photoreceptors and subsequent loss of vision. Two of the RP associated mutations were found in the CNGB1 gene that encodes the B subunit of the rod cyclic nucleotide-gated channel (CNGB1a). One of them (c.3444+1G.A) is located at the donor site of exon 32 and has been proposed to result in a frameshift and truncation of the last 28 aa of the corresponding protein. However, this ambiguous conclusion was not verified by experimental data. Recently, another study reported that the last 28 aa of CNGB1a harbor a motif required for the proper targeting of this subunit to rod photoreceptor outer segments. This suggests that defective targeting is the major cause for the RP phenotype in affected patients. Here, we investigated the splicing of c.3444+1G.A by exon trapping experiments and could demonstrate that instead of the proposed truncation of the last 28 aa this mutation leads to replacement of the last 170 aa of CNGB1a by 68 unrelated amino acids. The 170 aa deletion covers the complete distal C-terminus including the last 10 aa of an important alpha (aC) helix within the ligandbinding domain of CNGB1a. When expressed in a heterologous expression system the corresponding mutant full-length CNGB1a subunit was more susceptible to proteosomal degradation compared to the wild-type counterpart. In conclusion, our experimental data do not support the hypothesis proposed by the original study on the c.3444+1G.A mutation. Based on this, we suggest that apart from the defective targeting other mechanisms may be responsible for the RP phenotype i