Publications by authors named "Martin Biel"

160 Publications

Flavonoids increase melanin production and reduce proliferation, migration and invasion of melanoma cells by blocking endolysosomal/melanosomal TPC2.

Sci Rep 2021 Apr 19;11(1):8515. Epub 2021 Apr 19.

Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, Ludwig-Maximilians-University, Munich, Germany.

Two-pore channel 2 (TPC2) resides in endolysosomal membranes but also in lysosome-related organelles such as the melanin producing melanosomes. Gain-of-function polymorphisms in hTPC2 are associated with decreased melanin production and blond hair color. Vice versa genetic ablation of TPC2 increases melanin production. We show here an inverse correlation between melanin production and melanoma proliferation, migration, and invasion due to the dual activity of TPC2 in endolysosomes and melanosomes. Our results are supported by both genetic ablation and pharmacological inhibition of TPC2. Mechanistically, our data show that loss/block of TPC2 results in reduced protein levels of MITF, a major regulator of melanoma progression, but an increased activity of the melanin-generating enzyme tyrosinase. TPC2 inhibition thus provides a twofold benefit in melanoma prevention and treatment by increasing, through interference with tyrosinase activity, the synthesis of UV blocking melanin in melanosomes and by decreasing MITF-driven melanoma progression by increased GSK3β-mediated MITF degradation.
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http://dx.doi.org/10.1038/s41598-021-88196-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055690PMC
April 2021

Beyond pacemaking: HCN channels in sinoatrial node function.

Prog Biophys Mol Biol 2021 Mar 19. Epub 2021 Mar 19.

Center for Drug Research, Department of Pharmacy, Ludwig-Maximilians-Universität München, 81377, Munich, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, 80802, Munich, Germany. Electronic address:

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key proteins involved in the initiation and regulation of the heartbeat. Pacemaker cells within the sinoatrial node generate the electrical impulse that underlies the contraction of all atrial and ventricular cardiomyocytes. To generate a stable heart rhythm, it is necessary that the spontaneous activity of pacemaker cells is synchronized. Entrainment processes in the sinoatrial node create synchrony and also mediate heart rate regulation. In the past years it has become clear that the role of HCN channels goes beyond just pacemaking and that the channels play pivotal roles in these entrainment processes that coordinate and balance sinoatrial node network activity. Here, we review the role of HCN channels in the central pacemaker process and highlight new aspects of the contribution of HCN channels to stabilizing the electrical activity of the sinoatrial node network, especially during heart rate regulation by the autonomic nervous system.
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http://dx.doi.org/10.1016/j.pbiomolbio.2021.03.004DOI Listing
March 2021

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice.

J Vis Exp 2021 02 14(168). Epub 2021 Feb 14.

Center for Integrated Protein Science (CIPS-M) and Center for Drug Research, Department of Pharmacy, Ludwig-Maximilians-Universität München; German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance;

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR. Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.
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http://dx.doi.org/10.3791/62101DOI Listing
February 2021

Gene editing and synthetically accessible inhibitors reveal role for TPC2 in HCC cell proliferation and tumor growth.

Cell Chem Biol 2021 Feb 11. Epub 2021 Feb 11.

Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University Munich, 81377 Munich, Germany. Electronic address:

The role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of cancer cells in vitro, affects their energy metabolism, and successfully abrogates tumor growth in vivo. Concurrently, we have developed simplified analogs of the alkaloid tetrandrine as potent TPC2 inhibitors by screening a library of synthesized benzyltetrahydroisoquinoline derivatives. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against cancer cells and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing a TPC2 inhibitor with antitumor efficacy in mice. Hence, our study unveils TPC2 as valid target for cancer therapy and provides easily accessible tetrandrine analogs as a promising option for effective pharmacological interference.
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http://dx.doi.org/10.1016/j.chembiol.2021.01.023DOI Listing
February 2021

Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders.

EMBO Mol Med 2021 Apr 22;13(4):e13392. Epub 2021 Feb 22.

Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.

Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2-based peptide-display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high-level retinal transduction after intravitreal injection in mice, dogs and non-human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof-of-concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone-specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3 mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.
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http://dx.doi.org/10.15252/emmm.202013392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033523PMC
April 2021

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the -Deficient Mouse Model of Achromatopsia.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-University, 81377 Munich, Germany.

Mutations in the gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in KO mice. Virus-mediated knockdown or genetic ablation of in KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.
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http://dx.doi.org/10.3390/ijms22010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793084PMC
December 2020

TRPML2 is an osmo/mechanosensitive cation channel in endolysosomal organelles.

Sci Adv 2020 Nov 11;6(46). Epub 2020 Nov 11.

Walther Straub Institute of Pharmacology and Toxicology Faculty of Medicine, Ludwig-Maximilians-Universität, Munich, Germany.

Endolysosomes are dynamic, intracellular compartments, regulating their surface-to-volume ratios to counteract membrane swelling or shrinkage caused by osmotic challenges upon tubulation and vesiculation events. While osmosensitivity has been extensively described on the plasma membrane, the mechanisms underlying endolysosomal surface-to-volume ratio changes and identities of involved ion channels remain elusive. Endolysosomes mediate endocytosis, exocytosis, cargo transport, and sorting of material for recycling or degradation. We demonstrate the endolysosomal cation channel TRPML2 to be hypotonicity/mechanosensitive, a feature crucial to its involvement in fast-recycling processes of immune cells. We demonstrate that the phosphoinositide binding pocket is required for TRPML2 hypotonicity-sensitivity, as substitution of L314 completely abrogates hypotonicity-sensitivity. Last, the hypotonicity-insensitive TRPML2 mutant L314R slows down the fast recycling pathway, corroborating the functional importance of hypotonicity-sensitive TRPML2. Our results highlight TRPML2 as an accelerator of endolysosomal trafficking by virtue of its hypotonicity-sensitivity, with implications in immune cell surveillance and viral trafficking.
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http://dx.doi.org/10.1126/sciadv.abb5064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7673730PMC
November 2020

cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells.

Nat Commun 2020 11 3;11(1):5555. Epub 2020 Nov 3.

Hannover Medical School, Institute for Neurophysiology, 30625, Hannover, Germany.

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.
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http://dx.doi.org/10.1038/s41467-020-19304-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641277PMC
November 2020

A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation.

Sci Adv 2020 Aug 19;6(34):eaba5614. Epub 2020 Aug 19.

Center for Integrated Protein Science Munich CIPSM, Munich, Germany.

Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin () in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR-mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.
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http://dx.doi.org/10.1126/sciadv.aba5614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438099PMC
August 2020

Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation.

Mol Ther Nucleic Acids 2020 Sep 31;21:1050-1061. Epub 2020 Jul 31.

Center for Integrated Protein Science Munich CIPSM, Munich, Germany; Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany. Electronic address:

Mutations in CLRN1 cause Usher syndrome (USH) type III (USH3A), a disease characterized by progressive hearing impairment, retinitis pigmentosa, and vestibular dysfunction. Due to the lack of appropriate disease models, no efficient therapy for retinitis pigmentosa in USH patients exists so far. In addition, given the yet undefined functional role and expression of the different CLRN1 splice isoforms in the retina, non-causative therapies such as gene supplementation are unsuitable at this stage. In this study, we focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect. Surprisingly, however, no synergistic effect was detected when combining both methods. Finally, the injection of naked AONs into mice expressing the mutant CLRN1 minigene in the retina also led to a significant splice rescue. We propose that both AONs and CRISPR-Cas9 are suitable strategies to initiate advanced preclinical studies for treatment of USH3A patients.
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http://dx.doi.org/10.1016/j.omtn.2020.07.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452116PMC
September 2020

TPC1 deficiency or blockade augments systemic anaphylaxis and mast cell activity.

Proc Natl Acad Sci U S A 2020 07 13;117(30):18068-18078. Epub 2020 Jul 13.

Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, 80336 München, Germany;

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca) signals. Ca release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca uptake and filling of endoplasmic reticulum Ca stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.
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http://dx.doi.org/10.1073/pnas.1920122117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395440PMC
July 2020

Safety and Vision Outcomes of Subretinal Gene Therapy Targeting Cone Photoreceptors in Achromatopsia: A Nonrandomized Controlled Trial.

JAMA Ophthalmol 2020 06;138(6):643-651

Institute for Ophthalmic Research, Centre for Ophthalmology, University Hospital Tübingen, Tübingen, Germany.

Importance: Achromatopsia linked to variations in the CNGA3 gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available.

Objective: To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encoding CNGA3 (AAV8.CNGA3) in patients with CNGA3-linked achromatopsia.

Design, Setting, And Participants: This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants in CNGA3 were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018.

Intervention: Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011 total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017).

Main Outcomes And Measures: Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment.

Results: Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor-mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13; P = .006, 2-sided t test paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log; P = .003, 2-sided t test paired samples).

Conclusions And Relevance: Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains.

Trial Registration: ClinicalTrials.gov Identifier: NCT02610582.
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http://dx.doi.org/10.1001/jamaophthalmol.2020.1032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193523PMC
June 2020

Neuropathic and cAMP-induced pain behavior is ameliorated in mice lacking CNGB1.

Neuropharmacology 2020 07 6;171:108087. Epub 2020 Apr 6.

Institute of Pharmacology and Clinical Pharmacy, Goethe University, 60438, Frankfurt am Main, Germany.

Cyclic nucleotide-gated (CNG) channels, which are directly activated by cAMP and cGMP, have long been known to play a key role in retinal and olfactory signal transduction. Emerging evidence indicates that CNG channels are also involved in signaling pathways important for pain processing. Here, we found that the expression of the channel subunits CNGA2, CNGA3, CNGA4 and CNGB1 in dorsal root ganglia, and of CNGA2 in the spinal cord, is transiently altered after peripheral nerve injury in mice. Specifically, we show using in situ hybridization and quantitative real-time RT-PCR that CNG channels containing the CNGB1b subunit are localized to populations of sensory neurons and predominantly excitatory interneurons in the spinal dorsal horn. In CNGB1 knockout (CNGB1) mice, neuropathic pain behavior is considerably attenuated whereas inflammatory pain behavior is normal. Finally, we provide evidence to support CNGB1 as a downstream mediator of cAMP signaling in pain pathways. Altogether, our data suggest that CNGB1-positive CNG channels specifically contribute to neuropathic pain processing after peripheral nerve injury.
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http://dx.doi.org/10.1016/j.neuropharm.2020.108087DOI Listing
July 2020

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function.

Elife 2020 03 16;9. Epub 2020 Mar 16.

Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, Ludwig-Maximilians-Universität, Munich, Germany.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca-signals and non-selective cation currents, the other weaker Ca-signals and Na-selective currents. These properties were mirrored by the Ca-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.
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http://dx.doi.org/10.7554/eLife.54712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108868PMC
March 2020

Advancing Gene Therapy for PDE6A Retinitis Pigmentosa.

Adv Exp Med Biol 2019 ;1185:103-107

Center for Integrated Protein Science Munich (CIPSM) at the Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany.

Mutations in the gene encoding the phosphodiesterase 6 alpha subunit (PDE6A) account for 3-4% of autosomal recessive retinitis pigmentosa (RP), and currently no treatment is available. There are four animal models for PDE6A-RP: a dog with a frameshift truncating mutation (p.Asn616ThrfsTer39) and three mouse models with missense mutations (Val685Met, Asp562Trp, and Asp670Gly) showing a range of phenotype severities. Initial proof-of-concept gene augmentation studies in the Asp670Gly mouse model and dog model used a subretinally delivered adeno-associated virus serotype 8 with a 733 tyrosine capsid mutation delivering species-specific Pde6a cDNAs. These restored some rod-mediated function and preserved retinal structure. Subsequently, a translatable vector (AAV8 with a human rhodopsin promoter and human PDE6A cDNA) was tested in the dog and the Asp670Gly mouse model. In the dog, there was restoration of rod function, a robust rod-mediated ERG, and introduction of dim-light vision. Treatment improved morphology of the photoreceptor layer, and the retina was preserved in the treated region. In the Asp670Gly mouse, therapy also preserved photoreceptors with cone survival being reflected by maintenance of cone-mediated ERG responses. These studies are an important step toward a translatable therapy for PDE6A-RP.
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http://dx.doi.org/10.1007/978-3-030-27378-1_17DOI Listing
February 2020

Enigmatic rhodopsin mutation creates an exceptionally strong splice acceptor site.

Hum Mol Genet 2020 01;29(2):295-304

Center for Integrated Protein Science Munich (CIPSM), 81377 Munich, Germany.

The c.620 T > G mutation in rhodopsin found in the first mapped autosomal dominant retinitis pigmentosa (adRP) locus is associated with severe, early-onset RP. Intriguingly, another mutation affecting the same nucleotide (c.620 T > A) is related to a mild, late-onset RP. Assuming that both mutations are missense mutations (Met207Arg and Met207Lys) hampering the ligand-binding pocket, previous work addressed how they might differentially impair rhodopsin function. Here, we investigated the impact of both mutations at the mRNA and protein level in HEK293 cells and in the mouse retina. We show that, in contrast to c.620 T > A, c.620 T > G is a splicing mutation, which generates an exceptionally strong splice acceptor site (SAS) resulting in a 90 bp in-frame deletion and protein mislocalization in vitro and in vivo. Moreover, we identified the core element underlying the c.620 T > G SAS strength. Finally, we demonstrate that the c.620 T > G SAS is very flexible in branch point choice, which might explain its remarkable performance. Based on these results, we suggest that (i) point mutations should be routinely tested for mRNA splicing to avoid dispensable analysis of mutations on protein level, which do not naturally exist. (ii) Puzzling disease courses of mutations in other genes might also correlate with their effects on mRNA splicing. (iii) Flexibility in branch point choice might be another factor influencing the SAS strength. (iv) The core splice element identified in this study could be useful for biotechnological applications requiring effective SAS.
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http://dx.doi.org/10.1093/hmg/ddz291DOI Listing
January 2020

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes.

J Pharm Sci 2020 01 19;109(1):854-862. Epub 2019 Oct 19.

Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany; Center for Integrated Protein Science Munich (CiPSM), Munich, Germany; Department of Ophthalmology, Ludwig-Maximilians-Universität München, Munich, Germany. Electronic address:

Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (T), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the T of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid T. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.
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http://dx.doi.org/10.1016/j.xphs.2019.10.031DOI Listing
January 2020

HCN3 ion channels: roles in sensory neuronal excitability and pain.

J Physiol 2019 09 27;597(17):4661-4675. Epub 2019 Jul 27.

Wolfson Centre for Age-Related Research, King's College London, Guy's Campus, London, UK.

Key Points: HCN ion channels conducting the I current control the frequency of firing in peripheral sensory neurons signalling pain. Previous studies have demonstrated a major role for the HCN2 subunit in chronic pain but the potential involvement of HCN3 in pain has not been investigated. HCN3 was found to be widely expressed in all classes of sensory neurons (small, medium, large) where it contributes to I . HCN3 deletion increased the firing rate of medium but not small, sensory neurons. Pain sensitivity both acutely and following neuropathic injury was largely unaffected by HCN3 deletion, with the exception of a small decrease of mechanical hyperalgesia in response to a pinprick. We conclude that HCN3 plays little role in either acute or chronic pain sensation.

Abstract: HCN ion channels govern the firing rate of action potentials in the pacemaker region of the heart and in pain-sensitive (nociceptive) nerve fibres. Intracellular cAMP promotes activation of the HCN4 and HCN2 isoforms, whereas HCN1 and HCN3 are relatively insensitive to cAMP. HCN2 modulates action potential firing rate in nociceptive neurons and plays a critical role in all modes of inflammatory and neuropathic pain, although the role of HCN3 in nociceptive excitability and pain is less studied. Using antibody staining, we found that HCN3 is expressed in all classes of somatosensory neurons. In small nociceptive neurons, genetic deletion of HCN2 abolished the voltage shift of the I current carried by HCN isoforms following cAMP elevation, whereas the voltage shift was retained following deletion of HCN3, consistent with the sensitivity of HCN2 but not HCN3 to cAMP. Deletion of HCN3 had little effect on the evoked firing frequency in small neurons but enhanced the firing of medium-sized neurons, showing that HCN3 makes a significant contribution to the input resistance only in medium-sized neurons. Genetic deletion of HCN3 had no effect on acute thresholds to heat or mechanical stimuli in vivo and did not affect inflammatory pain measured with the formalin test. Nerve-injured HCN3 knockout mice exhibited similar levels of mechanical allodynia and thermal hyperalgesia to wild-type mice but reduced mechanical hyperalgesia in response to a pinprick. These results show that HCN3 makes some contribution to excitability, particularly in medium-sized neurons, although it has no major influence on acute or neuropathic pain processing.
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http://dx.doi.org/10.1113/JP278211DOI Listing
September 2019

Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures.

JCI Insight 2019 05 2;4(9). Epub 2019 May 2.

Department of Pharmacy, Center for Drug Research, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Munich, Germany.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.
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http://dx.doi.org/10.1172/jci.insight.126418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538325PMC
May 2019

Safety and Toxicology of Ocular Gene Therapy with Recombinant AAV Vector rAAV.hCNGA3 in Nonhuman Primates.

Hum Gene Ther Clin Dev 2019 06;30(2):50-56

1 University Eye Hospital Tübingen, University of Tübingen, Tübingen, Germany.

The purpose of this study was to examine the toxicity and side effects of a recombinant adeno-associated virus 8 (AAV8) vector, aimed to treat cyclic nucleotide gated channel alpha 3 ()-linked achromatopsia, after a single subretinal administration in cynomolgus macaques. Animals were followed in two studies: a 13-week study with 22 animals and a 28-day study with 12 animals. Both groups were divided into subgroups receiving either vehicle only, a low (1 × 10 vector genomes (vg)), or a high dose (1 × 10 vg) of rAAV.hCNGA3. In the 13-week study, an extra group received single high-dose intravitreal injections. Here we present the group results of the histological examinations carried out after necropsy from the 28-day study, the retinal functional (electroretinography) in the 13-week study, and clinical observations from both studies. Treatment-related adverse effects were not found, and parameter changes were mostly related to the surgical procedure. The treatment of achromatopsia with rAAV.hCNGA3 is therefore deemed safe to apply to humans.
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http://dx.doi.org/10.1089/humc.2018.188DOI Listing
June 2019

Selective agonist of TRPML2 reveals direct role in chemokine release from innate immune cells.

Elife 2018 11 27;7. Epub 2018 Nov 27.

Department of Pharmacology and Toxicology, Medical Faculty, Ludwig Maximilian University of Munich, Munich, Germany.

Cytokines and chemokines are produced and secreted by a broad range of immune cells including macrophages. Remarkably, little is known about how these inflammatory mediators are released from the various immune cells. Here, the endolysosomal cation channel TRPML2 is shown to play a direct role in chemokine trafficking and secretion from murine macrophages. To demonstrate acute and direct involvement of TRPML2 in these processes, the first isoform-selective TRPML2 channel agonist was generated, ML2-SA1. ML2-SA1 was not only found to directly stimulate release of the chemokine CCL2 from macrophages but also to stimulate macrophage migration, thus mimicking CCL2 function. Endogenous TRPML2 is expressed in early/recycling endosomes as demonstrated by endolysosomal patch-clamp experimentation and ML2-SA1 promotes trafficking through early/recycling endosomes, suggesting CCL2 being transported and secreted via this pathway. These data provide a direct link between TRPML2 activation, CCL2 release and stimulation of macrophage migration in the innate immune response.
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http://dx.doi.org/10.7554/eLife.39720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6257821PMC
November 2018

Accessory heterozygous mutations in cone photoreceptor CNGA3 exacerbate CNG channel-associated retinopathy.

J Clin Invest 2018 12 12;128(12):5663-5675. Epub 2018 Nov 12.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy.

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
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http://dx.doi.org/10.1172/JCI96098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264655PMC
December 2018

The protein interaction networks of mucolipins and two-pore channels.

Biochim Biophys Acta Mol Cell Res 2019 07 2;1866(7):1111-1123. Epub 2018 Nov 2.

Department of Pharmacology and Toxicology, Faculty of Medicine, University of Munich (LMU) Nussbaumstrasse 26, 80336 Munich. Electronic address:

Background: The endolysosomal, non-selective cation channels, two-pore channels (TPCs) and mucolipins (TRPMLs), regulate intracellular membrane dynamics and autophagy. While partially compensatory for each other, isoform-specific intracellular distribution, cell-type expression patterns, and regulatory mechanisms suggest different channel isoforms confer distinct properties to the cell.

Scope Of Review: Briefly, established TPC/TRPML functions and interaction partners ('interactomes') are discussed. Novel TRPML3 interactors are shown, and a meta-analysis of experimentally obtained channel interactomes conducted. Accordingly, interactomes are compared and contrasted, and subsequently described in detail for TPC1, TPC2, TRPML1, and TRPML3.

Major Conclusions: TPC interactomes are well-defined, encompassing intracellular membrane organisation proteins. TRPML interactomes are varied, encompassing cardiac contractility- and chaperone-mediated autophagy proteins, alongside regulators of intercellular signalling.

General Significance: Comprising recently proposed targets to treat cancers, infections, metabolic disease and neurodegeneration, the advancement of TPC/TRPML understanding is of considerable importance. This review proposes novel directions elucidating TPC/TRPML relevance in health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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http://dx.doi.org/10.1016/j.bbamcr.2018.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111325PMC
July 2019

Optimized Subretinal Injection Technique for Gene Therapy Approaches.

Methods Mol Biol 2019 ;1834:405-412

Division of Ocular Neurodegeneration, Institute for Ophthalmic Research, Centre for Ophthalmology, Eberhard Karls Universität Tübingen, Tübingen, Germany.

Gene therapy for inherited eye diseases requires local viral vector delivery by intraocular injection. Since large animal models are lacking for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The method described here aims to provide the details relevant to perform a transscleral pars plana virus-mediated gene transfer to achieve an optimized therapeutic effect in the small mouse eye.
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http://dx.doi.org/10.1007/978-1-4939-8669-9_26DOI Listing
April 2019

In Vitro Evaluation of AAV Vectors for Retinal Gene Therapy.

Methods Mol Biol 2019 ;1834:383-390

Department of Pharmacy-Center for Drug Research, Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Munich, Germany.

Gene therapy holds promise for treating previously untreatable retinal disorders. The most promising approaches use gene transfer vectors derived from adeno-associated virus (AAV) to supplement a gene function in the affected cell type. One example is gene therapy for achromatopsia which affects daylight vision. In this case, recombinant AAV (rAAV) vectors are being developed to specifically target cone photoreceptors. Development of rAAV vectors could be facilitated by the use of in vitro models. In this chapter we provide a protocol which utilizes mouse 661W cells, an in vitro model of cone photoreceptors for evaluation of the transduction efficacy of rAAV vectors.
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http://dx.doi.org/10.1007/978-1-4939-8669-9_24DOI Listing
April 2019

Development of Methodology and Study Protocol: Safety and Efficacy of a Single Subretinal Injection of rAAV.hCNGA3 in Patients with CNGA3-Linked Achromatopsia Investigated in an Exploratory Dose-Escalation Trial.

Hum Gene Ther Clin Dev 2018 09;29(3):121-131

3 STZ eyetrial at the Centre for Ophthalmology , Tuebingen, Germany .

Achromatopsia is an autosomal recessively inherited congenital defect characterized by a lack of cone photoreceptor function, leading to severely impaired vision. In this clinical study, achromatopsia patients were treated with a single subretinal injection of rAAV.hCNGA3 to restore cone function. The focus of this trial was on the safety of the treatment. After surgery, patients were monitored in eight extensive visits during the first year, followed by a 4-year follow-up period with annual visits. For essential complementation of the standard ophthalmological and systemic examinations, disease-specific methods were developed to assess the safety, efficacy, and patient-reported outcomes in this trial.
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http://dx.doi.org/10.1089/humc.2018.088DOI Listing
September 2018

Optogenetic Control of Neural Circuits in the Mongolian Gerbil.

Front Cell Neurosci 2018 24;12:111. Epub 2018 Apr 24.

Division of Neurobiology, Department Biology II, Biocenter, Ludwig Maximilian University of Munich, Munich, Germany.

The Mongolian gerbil () is widely used as a model organism for the human auditory system. Its hearing range is very similar to ours and it uses the same mechanisms for sound localization. The auditory circuits underlying these functions have been characterized. However, important mechanistic details are still under debate. To elucidate these issues, precise and reversible optogenetic manipulation of neuronal activity in this complex circuitry is required. However, genetic and genomic resources for the Mongolian gerbil are poorly developed. Here, we demonstrate a reliable gene delivery system using an AAV8(Y337F)-pseudotyped recombinant adeno-associated virus (AAV) 2-based vector in which the pan-neural human synapsin (hSyn) promoter drives neuron-specific expression of CatCH (Ca-permeable channelrhodopsin) or NpHR3.0 ( halorhodopsin). After stereotactic injection into the gerbil's auditory brainstem (medial nucleus of the trapezoid body, dorsal nucleus of the lateral lemniscus) and midbrain [inferior colliculus (IC)], we characterized CatCH- and/or NpHR3.0-transduced neurons in acute brain slices by means of whole-cell patch-clamp recordings. As the response properties of optogenetic tools strongly depend on neuronal biophysics, this parameterization is crucial for their application. In a proof-of-principle experiment in anesthetized gerbils, we observed strong suppression of sound-evoked neural responses in the dorsal nucleus of the lateral lemniscus (DNLL) and IC upon light activation of NpHR3.0. The successful validation of gene delivery and optogenetic tools in the Mongolian gerbil paves the way for future studies of the auditory circuits in this model system.
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http://dx.doi.org/10.3389/fncel.2018.00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928259PMC
April 2018

Humoral Immune Response After Intravitreal But Not After Subretinal AAV8 in Primates and Patients.

Invest Ophthalmol Vis Sci 2018 04;59(5):1910-1915

University Eye Hospital, University of Tübingen, Tübingen, Germany.

Purpose: To study longitudinal changes of anti-drug antibody (ADA) titers to recombinant adeno-associated virus serotype 8 (rAAV8) capsid epitopes in nonhuman primates (NHP) and patients.

Methods: Three groups of six NHP each received subretinal injections (high dose: 1 × 1012 vector genomes [vg], low dose: 1 × 1011 vg, or vehicle only). Four additional animals received intravitreal injections of the high dose (1 × 1012 vg). Three patients received 1 × 1010 vg as subretinal injections. ELISA quantified ADA levels at baseline and 1, 2, 3, 7, 28, and 90 days after surgery in NHP and at baseline and 1, 3, and 6 months after surgery in patients.

Results: Two out of 22 animals lacked ADA titers at baseline and developed low ADA titers toward the end of the study. Titers in the low-dose group stayed constant, while two of six animals from the high-dose group developed titers that rose beyond the range of the assay. All animals from the intravitreal control group showed a rise in ADA titer by day 7 that peaked at day 28. Preliminary data from the clinical trial (NCT02610582) show no humoral immune response in patients following subretinal delivery of 1 × 1010 vg.

Conclusions: No significant induction of ADA occurred in NHP when mimicking the clinical scenario of subretinal delivery with a clinical-grade rAAV8 and concomitant immunosuppression. Likewise, clinical data showed no humoral immune response in patients. In contrast, intravitreal delivery was associated with a substantial humoral immune response. Subretinal delivery might be superior to an intravitreal application regarding immunologic aspects.
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http://dx.doi.org/10.1167/iovs.17-22494DOI Listing
April 2018

Hif1a inactivation rescues photoreceptor degeneration induced by a chronic hypoxia-like stress.

Cell Death Differ 2018 12 17;25(12):2071-2085. Epub 2018 Apr 17.

Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zürich, Zürich, Switzerland.

Reduced choroidal blood flow and tissue changes in the ageing human eye impair oxygen delivery to photoreceptors and the retinal pigment epithelium. As a consequence, mild but chronic hypoxia may develop and disturb cell metabolism, function and ultimately survival, potentially contributing to retinal pathologies such as age-related macular degeneration (AMD). Here, we show that several hypoxia-inducible genes were expressed at higher levels in the aged human retina suggesting increased activity of hypoxia-inducible transcription factors (HIFs) during the physiological ageing process. To model chronically elevated HIF activity and investigate ensuing consequences for photoreceptors, we generated mice lacking von Hippel Lindau (VHL) protein in rods. This activated HIF transcription factors and led to a slowly progressing retinal degeneration in the ageing mouse retina. Importantly, this process depended mainly on HIF1 with only a minor contribution of HIF2. A gene therapy approach using AAV-mediated RNA interference through an anti-Hif1a shRNA significantly mitigated the degeneration suggesting a potential intervention strategy that may be applicable to human patients.
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http://dx.doi.org/10.1038/s41418-018-0094-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261999PMC
December 2018

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy.

Int J Mol Sci 2018 Mar 7;19(3). Epub 2018 Mar 7.

Center for Integrated Protein Science Munich (CIPSM), Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr, 5-13, 81377 Munich, Germany.

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.
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http://dx.doi.org/10.3390/ijms19030749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877610PMC
March 2018