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  • Colocalization of hyperpolarization-activated, cyclic nucleotide-gated channel subunits in rat retinal ganglion cells. 21456027

    The current-passing pore of mammalian hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels is formed by subunit isoforms denoted HCN1-4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons, and the current (I(h)) passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring I(h) in dissociated cells, and testing whether HCN1 and HCN4 proteins coimmunoprecipitate. Nearly half of the ganglion cells in whole-mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8-bromo-cAMP shifted the voltage sensitivity of I(h) less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Finally, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and I(h) activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native I(h) properties with the previously reported presence of HCN4 in these cells, and indicate that I(h) is biophysically similar in morphologically diverse retinal ganglion cells and differs from I(h) in rods, cones, and bipolar cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • HCN channels are expressed differentially in retinal bipolar cells and concentrated at synaptic terminals. 12786975

    Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels codetermine the integrative behaviour of neurons and shape their response to synaptic stimulation. We used immunohistochemistry and patch-clamp recording to study the composition and distribution of HCN channels in the rat retina. All four HCN channel isoforms (HCN1-4) are expressed differentially in the retina. In particular, different classes of bipolar cells have a different inventory of HCN channels. We found no evidence for the formation of heterooligomeric HCN channels. HCN channels are densely clustered at synaptic terminals of bipolar cells and photoreceptors. This suggests that HCN channels are involved in the control of transmitter release.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Differential expression of hyperpolarization-activated cyclic nucleotide-gated channel subunits during hippocampal development in the mouse. 25761792

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels help control the rhythmic activation of pacemaker neurons during brain development. However, little is known about the timing and cell type specificity of the expression of HCN isoforms during development of the hippocampus.Here we examined the developmental expression of the brain-enriched HCN1, HCN2, and HCN4 isoforms of HCN channels in mouse hippocampus from embryonic to postnatal stages. All these isoforms were expressed abundantly in the hippocampus at embryonic day 14.5 and postnatal day 0. Each HCN channel isoform showed subfield-specific expression within the hippocampus from postnatal day 7, and only HCN4 was found in glial cells in the stratum lacunosum moleculare at this developmental stage. At postnatal days 21 and 56, all HCN isoforms were strongly expressed in the stratum lacunosum moleculare and the stratum pyramidale of the Cornu Ammonis (CA), as well as in the hilus of the dentate gyrus, but not in the subgranular zone. Furthermore, the immunolabeling for all these isoforms was colocalized with parvalbumin immunolabeling in interneurons of the CA field and in the dentate gyrus.Our mapping data showing the temporal and spatial changes in the expression of HCN channels suggest that HCN1, HCN2, and HCN4 subunits may have distinct physiological roles in the developing hippocampus.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1572
    Nombre del producto:
    Anti-Parvalbumin Antibody

  • Endoplasmic reticulum stress-associated cone photoreceptor degeneration in cyclic nucleotide-gated channel deficiency. 22493484

    Cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 account for greater than 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-IP(3)R, 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.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-540
    Nombre del producto:
    Anti-acetyl-Histone H3 (Lys36) Antibody

  • Hyperpolarization-activated cation channels are expressed in rat hypothalamic gonadotropin-releasing hormone (GnRH) neurons and immortalized GnRH neurons. 16879992

    OBJECTIVES: The current research was conducted to determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are expressed in gonadotropin-releasing hormone (GnRH) neurons in the female rat hypothalamus and immortalized GnRH neurons (GT1-7 cells). METHODS: Double-label fluorescence immunohistochemistry was used to colocalize HCN1-4 channels and GnRH in GnRH neurons in the female rat hypothalamus. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were used to analyze HCN channel gene expression in GT1-7 cells. RESULTS: Double-label fluorescence immunohistochemistry showed that 43% of hypothalamic GnRH neurons immunostained for HCN2 and 90% of GnRH neurons immunostained for HCN3. RT-PCR and Western blot showed expression of all four HCN channel subunits in GT1-7 cells. Double-label immunocytochemistry showed cytoplasmic immunostaining of HCN2 and HCN3 in GT1-7 cells. CONCLUSIONS: This study demonstrates for the first time that HCN channels are expressed in GnRH neurons in the rat hypothalamus and GT1-7 cells. Our research supports the hypothesis that HCN channels may be involved in electrical bursting activity and pulsatile GnRH secretion in endogenous GnRH neurons and GT1-7 cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5456

  • Quantitative analysis and subcellular distribution of mRNA and protein expression of the hyperpolarization-activated cyclic nucleotide-gated channels throughout developme ... 16648453

    The properties of the hyperpolarization-activated current (I(h)) and its roles in hippocampal network function evolve radically during development. Because I(h) is conducted by the hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, we tested the hypothesis that understanding the quantitative developmental profiles of HCN1, HCN2, and HCN4 expression, and the isoform- and age-specific progression of their subcellular distribution, should shed light on the established modifications of the properties of I(h) throughout development. Combined quantitative in situ hybridization, regional western blots, and high-resolution, dual-label immunocytochemistry revealed striking and novel information about the expression and distribution of the HCN channel isoforms in the developing hippocampal formation. In cornus ammon 1 (CA) pyramidal cell layer, a robust increase of HCN1 mRNA and protein expression occurred with age, with reciprocal reduction of HCN4 and relatively stable HCN2 levels. These distinct expression patterns raised the contribution of HCN1 to the total HCN channel pool from 33% to 65% consonant with acceleration and reduced cyclic adenosine mono phosphate (cAMP) sensitivity of I(h) in this region with age. In CA3, strong expression of HCN1 already neonatally supports the recently established role of this conductance in neonatal, age-specific, hippocampal oscillations (giant depolarizing potentials). Notably, HCN1 channels were present and probably transported to dendritic compartments already on postnatal day (P) 2, whereas HCN2 channel protein was not evident in dendrites for the first 2 weeks of life. HCN2 mRNA and protein expression remained fairly constant subsequent to the first week of life in all hippocampal subfields examined, whereas HCN4 mRNA and protein expression declined after maximal neonatal expression, so that the contribution of this isoform to the total HCN channel pool dropped from 43% (CA1) and 34% (CA3) on P11 to 8% (CA1) and 19% (CA3) on P90. Interneuronal expression of all HCN channel isoforms in stratum pyramidale was robust in parvalbumin-but not in cholecystokinin-expressing populations and with a subunit-specific subcellular distribution. Taken together, these data suggest that early in life, HCN4 may contribute significantly to the functions of I(h) in specific hippocampal regions. In addition, these evolving, differential quantitative, and subcellular expression patterns of the HCN channel isoforms support age-specific properties and functions of I(h) within the developing hippocampal formation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Disease-associated mutations in CNGB3 promote cytotoxicity in photoreceptor-derived cells. 23805033

    To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells.Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3',5'-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes.Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium.These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1501
    Nombre del producto:
    Anti-Actin Antibody, clone C4

  • Hyperpolarization-activated and cyclic nucleotide-gated channels are differentially expressed in juxtaglomerular cells in the olfactory bulb of mice. 20140458

    In the olfactory bulb, input from olfactory receptor neurons is processed by neuronal networks before it is relayed to higher brain regions. In many neurons, hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels generate and control oscillations of the membrane potential. Oscillations also appear crucial for information processing in the olfactory bulb. Four channel isoforms exist (HCN1-HCN4) that can form homo- or heteromers. Here, we describe the expression pattern of HCN isoforms in the olfactory bulb of mice by using a novel and comprehensive set of antibodies against all four isoforms. HCN isoforms are abundantly expressed in the olfactory bulb. HCN channels can be detected in most cell populations identified by commonly used marker antibodies. The combination of staining with marker and HCN antibodies has revealed at least 17 different staining patterns in juxtaglomerular cells. Furthermore, HCN isoforms give rise to an unexpected wealth of co-expression patterns but are rarely expressed in the same combination and at the same level in two given cell populations. Therefore, heteromeric HCN channels may exist in several cell populations in vivo. Our results suggest that HCN channels play an important role in olfactory information processing. The staining patterns are consistent with the possibility that both homomeric and heteromeric HCN channels are involved in oscillations of the membrane potential of juxtaglomerular cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1550

  • Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption. 21559843

    Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 μM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3132