Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed Ifor Ih, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel ...family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, Ifin cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4-/-mice, cardiac cells with "primitive" pacemaker action potentials could be found. However, cardiac cells with "mature" pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node.
Endoplasmic reticulum (ER) stress and mislocalization of improperly folded proteins have been shown to contribute to photoreceptor death in models of inherited retinal degenerative diseases. In ...particular, mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model for achromatopsia, display both early-onset ER stress and opsin mistrafficking. By 2 weeks of age, these mice show elevated signaling from all three arms of the ER-stress pathway, and by 1 month, cone opsin is improperly distributed away from its normal outer segment location to other retinal layers. This work investigated the role of Ca2+-release channels in ER stress, protein mislocalization, and cone death in a mouse model of CNG-channel deficiency. We examined whether preservation of luminal Ca2+ stores through pharmacological and genetic suppression of ER Ca2+ efflux protects cones by attenuating ER stress. We demonstrated that the inhibition of ER Ca2+-efflux channels reduced all three arms of ER-stress signaling while improving opsin trafficking to cone outer segments and decreasing cone death by 20–35%. Cone-specific gene deletion of the inositol-1,4,5-trisphosphate receptor type I (IP3R1) also significantly increased cone density in the CNG-channel-deficient mice, suggesting that IP3R1 signaling contributes to Ca2+ homeostasis and cone survival. Consistent with the important contribution of organellar Ca2+ signaling in this achromatopsia mouse model, significant differences in dynamic intraorganellar Ca2+ levels were detected in CNG-channel-deficient cones. These results thus identify a novel molecular link between Ca2+ homeostasis and cone degeneration, thereby revealing novel therapeutic targets to preserve cones in inherited retinal degenerative diseases.
Abstract
Tet3 is the main α-ketoglutarate (αKG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high ...levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How αKG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into αKG in an NAD
+
-dependent manner, is redirected to the nucleus by the αKG-consumer protein Tet3, suggesting on-site production of αKG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of αKG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity.
Infectious diseases and cancer are among the key medical challenges that humankind is facing today. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial ...role in the pathology of both groups of diseases. The development of advanced patch-clamp technologies has allowed us to directly characterize ion fluxes through endolysosomal ion channels in their native environments. Endolysosomes are essential organelles for intracellular transport, digestion and metabolism, and maintenance of homeostasis. The endolysosomal ion channels regulate the function of the endolysosomal system through four basic mechanisms: calcium release, control of membrane potential, pH change, and osmolarity regulation. In this review, we put particular emphasis on the endolysosomal cation channels, including TPC2 and TRPML2, which are particularly important in monocyte function. We discuss existing endogenous and synthetic ligands of these channels and summarize current knowledge of their impact on channel activity and function in different cell types. Moreover, we summarize recent findings on the importance of TPC2 and TRPML2 channels as potential drug targets for the prevention and treatment of the emerging infectious diseases and cancer.
Display omitted
•Biophysical properties of TPC2 and TRPML2 were characterized in whole-endolysosome recordings.•TPC2 and TRPML2 play critical roles in cancer and infectious diseases.•The roles of TPC2 and TRPML2 in monocytic lineage cells.•TPC2 and TRPML2 as novel drug targets in precision medicine.
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.
Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and requires surgical implantation of electric pacemaker devices. Frequently, SND ...occurs in heart failure and hypertension, conditions that lead to electric instability of the heart. Although the pathologies of acquired SND have been studied extensively, little is known about the molecular and cellular mechanisms that cause congenital SND.
Here, we show that the HCN1 protein is highly expressed in the sinoatrial node and is colocalized with HCN4, the main sinoatrial pacemaker channel isoform. To characterize the cardiac phenotype of HCN1-deficient mice, a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparation, telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology was performed. On the basis of these experiments we demonstrate that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output.
We propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.
Vision originates in rods and cones at the outer retina. Already at these early stages, diverse processing schemes shape and enhance image information to permit perception over a wide range of ...lighting conditions. In this work, we address the role of hyperpolarization-activated and cyclic nucleotide-gated channels 1 (HCN1) in rod photoreceptors for the enhancement of rod system responsivity under conditions of light exposure.
To isolate HCN1 channel actions in rod system responses, we generated double mutant mice by crossbreeding Hcn1-/- mice with Cnga3-/- mice in which cones are non-functional. Retinal function in the resulting Hcn1-/- Cnga3-/- animals was followed by means of electroretinography (ERG) up to the age of four month. Retinal imaging via scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) was also performed to exclude potential morphological alterations.
This study on Hcn1-/- Cnga3-/- mutant mice complements our previous work on HCN1 channel function in the retina. We show here in a functional rod-only setting that rod responses following bright light exposure terminate without the counteraction of HCN channels much later than normal. The resulting sustained signal elevation does saturate the retinal network due to an intensity-dependent reduction in the dynamic range. In addition, the lack of rapid adaptational feedback modulation of rod photoreceptor output via HCN1 in this double mutant limits the ability to follow repetitive (flicker) stimuli, particularly under mesopic conditions.
This work corroborates the hypothesis that, in the absence of HCN1-mediated feedback, the amplitude of rod signals remains at high levels for a prolonged period of time, leading to saturation of the retinal pathways. Our results demonstrate the importance of HCN1 channels for regular vision.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone ...dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca2+ channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3−/−/Nrl−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca2+ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.
Background: Cone photoreceptors undergo endoplasmic reticulum stress-associated apoptosis in CNG channel deficiency.
Results: Suppressing cGMP/PKG signaling enhances inositol 1,4,5-trisphosphate receptor 1 (IP3R1) phosphorylation and inhibits endoplasmic reticulum stress and cone death.
Conclusion: cGMP/PKG signaling regulates IP3R1 activity and promotes endoplasmic reticulum stress in CNG channel deficiency.
Significance: Understanding of the mechanism(s) of photoreceptor degeneration is essential for therapeutic strategy development.
The mammalian main olfactory epithelium (MOE) recognizes and transduces olfactory cues through a G protein-coupled, cAMP-dependent signaling cascade. Additional chemosensory transduction mechanisms ...have been suggested but remain controversial. We show that a subset of MOE neurons expressing the orphan receptor guanylyl cyclase GC-D and the cyclic nucleotide-gated channel subunit CNGA3 employ an excitatory cGMP-dependent transduction mechanism for chemodetection. By combining gene targeting of Gucy2d, which encodes GC-D, with patch clamp recording and confocal Ca²⁺ imaging from single dendritic knobs in situ, we find that GC-D cells recognize the peptide hormones uroguanylin and guanylin as well as natural urine stimuli. These molecules stimulate an excitatory, cGMP-dependent signaling cascade that increases intracellular Ca²⁺ and action potential firing. Responses are eliminated in both Gucy2d- and Cnga3-null mice, demonstrating the essential role of GC-D and CNGA3 in the transduction of these molecules. The sensitive and selective detection of two important natriuretic peptides by the GC-D neurons suggests the possibility that these cells contribute to the maintenance of salt and water homeostasis or the detection of cues related to hunger, satiety, or thirst.
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.
Photoreceptors undergo degeneration when phototransduction is impaired.
The endoplasmic reticulum stress markers and processing of the associated caspases are elevated in retinas with cone photoreceptor CNG channel deficiency.
The endoplasmic reticulum stress-associated apoptotic pathways play a crucial role in cone degeneration.
Understanding of the mechanism(s) of photoreceptor degeneration is essential for development of therapeutic strategies.
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