To characterize molecular and cellular changes in the mouse retina caused by the genetic deletion of the cone cyclic nucleotide-gated channel (CNG) subunit CNGA3.
Retinas of wild-type and ...CNGA3-deficient (CNGA3(-/-)) mice from 9 days up to 22 months of age were analyzed by immunohistochemistry, electron microscopy, and molecular biological methods.
CNGA3(-/-) cones failed to transport opsins into outer segments, downregulated various proteins of the phototransduction cascade, and induced apoptotic death. Loss of CNGA3 did not affect the transcription of cone-specific genes. Cone degeneration was evident from the second postnatal week on, and it proceeded significantly faster in the ventral than in the dorsal part of the retina. Ventral cones were completely missing after the third postnatal month, whereas residual dorsal cones were present, even in 22-month-old knockout mice. CNGA3(-/-) cone somata exhibited profoundly delayed migration during postnatal development. At the time of eye opening, most CNGA3(-/-) cones had displaced somata localized close to or in the outer plexiform layer. These cones lacked the characteristic synaptic pedicle, but revealed synapselike contacts to second-order neurons at their somata. At later stages, most of the surviving CNGA3(-/-) cones had correctly located somata and morphologically normal synapses.
The loss of CNGA3 impairs the targeting of cone opsins and the expression of other visual cascade proteins. In addition, CNGA3 appears to be essential for normal postnatal migration of cone somata. After loss of cone outer segment proteins, CNGA3(-/-) cones induce apoptotic cell death.
Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of ...subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.
Two types of photoreceptors, rods and cones, coexist in the vertebrate retina. An in-depth analysis of the retinal circuitry that transmits rod and cone signals has been hampered by the presence of ...intimate physical and functional connections between rod and cone pathways. By deleting the cyclic nucleotide-gated channel CNG3 we have generated a mouse lacking any cone-mediated photoresponse. In contrast, the rod pathway is completely intact in CNG3-deficient mice. The functional loss of cone function correlates with a progressive degeneration of cone photoreceptors but not of other retinal cell types. CNG3-deficient mice provide an animal model to dissect unequivocally the contribution of rod and cone pathways for normal retinal function.
Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in ...brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.
Abstract
In der Grundlagenforschung wird die Testung heterogener Katalysatoren normalerweise mit sehr kleinen Probenmengen durchgeführt. Eine Übertragung der ermittelten Ergebnisse hinsichtlich ...Produktivität und Standzeit auf den technischen Maßstab ist nur sehr eingeschränkt möglich. Es wurde ein neues praxisnahes Testsystem für heterogene Katalysatoren entwickelt. Es kann sowohl in Festbett‐Fahrweise als auch in Slurry‐Fahrweise in einem weiten Temperatur‐ und Druckbereich betrieben werden. Das System wurde für die Synthese höherer Alkohole und die einstufige Dimethylether‐Synthese erfolgreich eingesetzt.
Abstract
In fundamental heterogeneous catalyst development, testing is usually performed with rather small amounts of catalyst. Transferring the results in terms of performance and catalyst lifetime to industrial relevant pilot‐scale is hardly feasible. Thus, a new modular test system has been developed which allows large‐scale testing of heterogeneous catalysts in fixed‐bed or slurry mode, enabling a wide range of test conditions with regard to particle size, gas composition, pressure, and reaction temperature. The test system has been used for higher alcohol synthesis and one‐step dimethyl ether synthesis.
In der Grundlagenforschung wird die Testung heterogener Katalysatoren normalerweise mit sehr kleinen Probenmengen durchgeführt. Eine Übertragung der ermittelten Ergebnisse hinsichtlich Produktivität ...und Standzeit auf den technischen Maßstab ist nur sehr eingeschränkt möglich. Es wurde ein neues praxisnahes Testsystem für heterogene Katalysatoren entwickelt. Es kann sowohl in Festbett‐Fahrweise als auch in Slurry‐Fahrweise in einem weiten Temperatur‐ und Druckbereich betrieben werden. Das System wurde für die Synthese höherer Alkohole und die einstufige Dimethylether‐Synthese erfolgreich eingesetzt.
In fundamental heterogeneous catalyst development, testing is usually performed with rather small amounts of catalyst. Transferring the results in terms of performance and catalyst lifetime to industrial relevant pilot‐scale is hardly feasible. Thus, a new modular test system has been developed which allows large‐scale testing of heterogeneous catalysts in fixed‐bed or slurry mode, enabling a wide range of test conditions with regard to particle size, gas composition, pressure, and reaction temperature. The test system has been used for higher alcohol synthesis and one‐step dimethyl ether synthesis.
Vorgestellt wird ein neues System zur praxisnahen Testung von heterogenen Katalysatoren. Das System soll die Lücke zwischen der Katalysatortestung im Labormaßstab und im technischen Maßstab schließen. Im Festbett‐ und im Slurry‐Reaktor wurden Katalysatoren für die Synthesegaskonversion zu Alkoholen und Dimethylether getestet.
Cyclic nucleotide-gated (CNG) channels are important mediators in the transduction pathways of rod and cone photoreceptors. Native CNG channels are heterotetramers composed of homologous A and B ...subunits. In heterologous expression systems, B subunits alone cannot form functional CNG channels, but they confer a number of channel properties when coexpressed with A subunits. To investigate the importance of the CNGB subunits in vivo, we deleted the CNGB1 gene in mice. In the absence of CNGB1, only trace amounts of the CNGA1 subunit were found on the rod outer segment. As a consequence, the vast majority of isolated rod photoreceptors in mice lacking CNGB1 (CNGB1-/-) failed to respond to light. In electroretinograms (ERGs), CNGB1-/- mice showed no rod-mediated responses. The rods also showed a slow-progressing degeneration caused by apoptotic death and concurred by retinal gliosis. Cones were primarily unaffected and showed normal ERG responses up to 6 months, but they started to degenerate in later stages. At the age of approximately 1 year, CNGB1-/- animals were devoid of both rods and cones. Our results show that CNGB1 is a crucial determinant of native CNG channel targeting. As a result of the lack of rod CNG channels, CNGB1-/- mice develop a retinal degeneration that resembles human retinitis pigmentosa.
Cyclic nucleotide-gated (CNG) channels play a key role in olfactory and visual transduction. Native CNG channels are heteromeric complexes consisting of the principal alpha subunits (CNG1-3), which ...can form functional channels by themselves, and the modulatory beta subunits (CNG4-5). The individual alpha and beta subunits that combine to form the CNG channels in rod photoreceptors (CNG1 + CNG4) and olfactory neurons (CNG2 + CNG4 + CNG5) have been characterized. In contrast, only an alpha subunit (CNG3) has been identified so far in cone photoreceptors. Here we report the molecular cloning of a new CNG channel subunit (CNG6) from mouse retina. The cDNA of CNG6 encodes a peptide of 694 amino acids with a predicted molecular weight of 80 kDa. Among the CNG channel subunits, CNG6 has the highest overall similarity to the CNG4 beta subunit (47% sequence identity). CNG6 transcripts are present in a small subset of retinal photoreceptor cells and also in testis. Heterologous expression of CNG6 in human embryonic kidney 293 cells did not lead to detectable currents. However, when coexpressed with the cone photoreceptor alpha subunit, CNG6 induced a flickering channel gating, weakened the outward rectification in the presence of extracellular Ca(2+), increased the sensitivity for L-cis diltiazem, and enhanced the cAMP efficacy of the channel. Taken together, the data indicate that CNG6 represents a new CNG channel beta subunit that may associate with the CNG3 alpha subunit to form the native cone channel.
Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of ...subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1
−/−
) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1
−/−
mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1
−/−
mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca
2+
-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1
−/−
mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function
in vivo
. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.
To study the electrophysiological and pharmacological properties of the L-type Ca(2+) channel (LTCC) Ca(v)1.4alpha1 (alpha1F) subunit from mouse retina and assess their contributions to the native ...retinal channel.
The full-length cDNA of Ca(v)1.4alpha1 was cloned from murine retina in an RT-PCR approach. Ca(v)1.4alpha1 was expressed alone or together with the auxiliary alpha2delta1 and beta2a or beta3 subunits in HEK293 cells. The electrophysiological and pharmacological characteristics of L-type Ca(2+) and Ba(2+) inward currents (I(Ca) and I(Ba)) induced by Ca(v)1.4alpha1 were determined by the whole-cell configuration of the patch-clamp method and compared with currents induced by the cardiac and smooth muscle-type Ca(v)1.2alpha1 (alpha1C) channel.
Ca(v)1.4alpha1-mediated I(Ba) was observed only when the alpha2delta1 and beta subunits were coexpressed. Current densities were approximately two times higher with beta2a than with beta3. I(Ba) activated faster and revealed much slower time-dependent inactivation than I(Ba) induced by Ca(v)1.2alpha1. Unlike in Ca(v)1.2alpha1, inactivation was not accelerated with Ca(2+) as the charge carrier, indicating the absence of Ca(2+)-dependent inactivation in Ca(v)1.4alpha1. Ca(v)1.4alpha1 exhibited voltage-dependent inactivation. The dihydropyridine (DHP) antagonist isradipine blocked Ca(v)1.4alpha1 with approximately 20-fold lower sensitivity than Ca(v)1.2alpha1. The agonistic DHP BayK 8644 stimulated maximum I(Ba) approximately sixfold. Ca(v)1.4alpha1 revealed only moderate sensitivities to L- and D-cis-diltiazem, with IC(50) in the micromolar range. Both enantiomers unexpectedly blocked Ca(v)1.4alpha1 with almost equal IC(50).
The data indicate that Ca(v)1.4alpha1 subunit constitutes the major molecular correlate of retinal L-type Ca(2+) current. Its intrinsic biophysical properties, in particular its unique inactivation properties, enable Ca(v)1.4alpha1 to provide a sustained I(Ca) over a voltage range such as required for tonic glutamate release at the photoreceptor synapse.
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