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U B Kaupp

Publications and source records attributed to U B Kaupp.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cyclic nucleotide-gated channels on the flagellum control Ca2+ entry into sperm.

Cyclic nucleotide-gated (CNG) channels are key elements of cGMP- and cAMP-signaling pathways in vertebrate photoreceptor cells and in olfactory sensory neurons, respectively. These channels form heterooligomeric complexes composed of at least two distinct subunits (alpha and beta). The alpha subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of beta subunits in testis. The alpha and beta subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the alpha subunit is observed along the entire flagellum, whereas the short beta subunit is restricted to the principal piece of the flagellum. These findings suggest that different forms of CNG channels coexist in the flagellum. Confocal microscopy in conjunction with the Ca2+ indicator Fluo-3 shows that the CNG channels serve as a Ca2+ entry pathway that responds more sensitively to cGMP than to cAMP. Assuming that CNG channel subtypes differ in their Ca2+ permeability, dissimilar localization of alpha and beta subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.

Amino Acid Sequence↗
PubMed1998

Molecular identification of a hyperpolarization-activated channel in sea urchin sperm.

Sea urchin eggs attract sperm through chemotactic peptides, which evoke complex changes in membrane voltage and in the concentrations of cyclic AMP, cyclic GMP and Ca2+ ions The intracellular signalling pathways and their cellular targets are largely unknown. We have now cloned, from sea urchin testis, the complementary DNA encoding a channel polypeptide, SPIH. Functional expression of SPIH gives rise to weakly K+-selective hyperpolarization-activated channels, whose activity is enhanced by the direct action of cAMP. Thus, SPIH is under the dual control of voltage and cAMP. The SPIH channel, which is confined to the sperm flagellum, may be involved in the control of flagellar beating. SPIH currents exhibit all the hallmarks of hyperpolarization-activated currents (Ih), which participate in the rhythmic firing of central neurons, control pacemaking in the heart, and curtail saturation by bright light in retinal photoreceptors. Because of their sequence and functional properties, Ih channels form a class of their own within the superfamily of voltage-gated and cyclic-nucleotide-gated channels.

Amino Acid Sequence↗
PubMed1998

Calmodulin controls the rod photoreceptor CNG channel through an unconventional binding site in the N-terminus of the beta-subunit.

Calmodulin (CaM) controls the activity of the rod cGMP-gated ion channel by decreasing the apparent cGMP affinity. We have examined the mechanism of this modulation using electrophysiological and biochemical techniques. Heteromeric channels, consisting of alpha- and beta-subunits, display a high CaM sensitivity (EC50 </=5 nM) similar to the native channel. Using surface plasmon resonance spectroscopy, we identified two unconventional CaM-binding sites (CaM1 and CaM2), one in each of the N- and the C-terminal regions of the beta-subunit. Ca2+ co-operatively stimulates binding of CaM to these sites exactly within the range of [Ca2+] occurring during a light response. Deletion of the N-terminal CaM1 site results in channels that are no longer CaM-sensitive, whereas deletion of CaM2 has only minor effects. We discuss different models to explain the high-affinity binding of CaM.

Amino Acid Sequence↗
PubMed1998

[Signal transduction in photoreceptor cells].

Vertebrate photoreceptors respond to light with a brief hyperpolarization of their membrane potential. In the dark, photoreceptors are depolarized by cation influx through channels in the plasma membrane which are kept open by the second messenger cGMP. Light absorption activates an enzyme cascade that hydrolytically destroys cGMP, resulting in channel closure and hyperpolarization of the membrane. In addition, processes are initiated that allow photoreceptors to adapt their sensitivity to the ambient illumination. Although these adaptational mechanisms are less well understood, it is clear that they are strongly controlled by the intracellular Ca2+ concentration. This review describes our present knowledge about the signal transduction and its fine tuning by a complex network of Ca(2+)-mediated processes in vertebrate photoreceptors.

Animals↗
PubMed1998

Novel caged compounds of hydrolysis-resistant 8-Br-cAMP and 8-Br-cGMP: photolabile NPE esters.

The application of the 1-(2-nitrophenyl)ethyl (NPE) moiety as a photolabile ligand for the release of hydrolysis-resistant 8-Br-cAMP and 8-Br-cGMP was examined. NPE-caged 8-Br-cAMP and 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light. The synthesis procedure resulted in diastereoisomeric mixtures, which were chromatographically separated into the axial and equatorial isomers of NPE-caged 8-Br-cAMP and 8-Br-cGMP. The hydrolytic stability, solubility and photochemical properties of these derivatives were compared to the previously reported 4.5-dimethoxy-2-nitrobenzyl (DMNB) compounds. We found that the axial isomers of NPE-caged 8-Br-cAMP and 8-Br-cGMP had a considerably better solvolytic stability than the respective equatorial isomers as well as the DMNB-caged derivatives. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Characterization of ether-à-go-go channels present in photoreceptors reveals similarity to IKx, a K+ current in rod inner segments.

In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels.

Amino Acid Sequence↗
PubMed1997

Cloning, chromosomal localization and functional expression of the gene encoding the alpha-subunit of the cGMP-gated channel in human cone photoreceptors.

Cyclic nucleotide-gated (CNG) ion channels serve as final targets of signal transduction in vertebrate photoreceptors. While the basic mechanisms of phototransduction are similar in rod and cone photoreceptors, both cell types express distinct sets of components of the transduction pathway. We report here the cloning of the cDNA encoding the alpha-subunit of the cGMP-gated channel of human cone photoreceptors. The open reading frame predicts a polypeptide of 694 amino acid residues with conserved functional parts and amino acid positions typical for the alpha-subunit of CNG-channels. Heterologous expression of the cDNA in Xenopus oocytes gave rise to cGMP-gated channel activity. Antiserum directed against the C-terminus of the bovine cone CNG channel alpha-subunit crossreacted specifically with the heterologously expressed polypeptide and stained cone photoreceptors and weakly also the outer plexiform layer in human retinal sections. Northern blot analysis detected a prominent mRNA species of approximately 3.8 kb in human retina. The entire gene spans approximately 30 kb of genomic sequence and is located on the pericentric band q11.2 of human chromosome 2. The gene is composed of seven exons, with introns located at positions which are preserved with respect to the human rod gene, indicating a common ancestral gene structure. RT-PCR analysis gave no evidence for alternatively spliced transcripts.

Animals↗
PubMed1996

Two alternatively spliced forms of the cGMP-gated channel alpha-subunit from cone photoreceptor are expressed in the chick pineal organ.

Light sensitivity of the pineal has been retained in most vertebrates, except mammals. Retinal photoreceptors and pinealocytes share common components of light-dependent signaling pathways. In particular, an ion channel gated by cGMP has been electrophysiologically identified in chick pinealocytes; however, the physiological function of a light-sensitive enzyme cascade is not known, and primary structures of only a few pineal components have been determined. By PCR analysis and cloning of the respective cDNA, we show that the chick pineal expresses the alpha-subunit of the cyclic nucleotide-gated (CNG) channel of rod photoreceptors and two short forms of the cone CNG channel. Analysis of the chick cone CNG channel gene reveals that these forms are produced by alternative splicing, which removes either one or two exons from the transcript. The shorter splice variant is functional when heterologously expressed, and it is approximately twofold more sensitive to activation by cGMP than the cone CNG channel. The chick cone CNG channel and the pineal splice form are both modulated by Ca2+/calmodulin (CaM). The CaM sensitivity might be mediated by a putative CaM-binding site in an N-terminal segment encoded by exon 4. This exon is missing in the gene for the rod CNG channel alpha-subunit. Pineal CNG channels are candidates for receptor-mediated Ca2+ entry into pinealocytes and may be an important element of signaling pathways that control the light response and secretion of the pineal hormone melatonin.

Alternative Splicing↗
PubMed1996

Caged compounds of hydrolysis-resistant analogues of cAMP and cGMP: synthesis and application to cyclic nucleotide-gated channels.

Photolabile compounds which rapidly release cAMP or cGMP after photolysis are widely used for in situ studies of signaling pathways inside cells. We synthesized two novel caged compounds, 4,5-dimethoxy-2-nitrobenzyl 8-Br-cAMP (caged 8-Br-cAMP) and 4,5-dimethoxy-2-nitrobenzyl 8-Br-cGMP caged 8-BR-cGMP), which respectively release the hydrolysis-resistant analogues 8-Br-cAMP and 8-Br-cGMP. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons. The synthesis procedure resulted in diastereomeric mixtures which were chromatographically separated into the axial and equatorial isomers of caged 8-BR-cAMP and of caged 8-BR-cGMP. The axial isomers which have a higher solubility and better solvolytic stability than the equatorial forms were used for experiments with CNG channels. Flashes of UV light produced steps in the concentration of 8-Br-cGMP which activated currents through CNG channels. Concentration steps inside the cell could be calibrated precisely using the relation between the ligand concentration and the normalized current. Similar results were obtained with caged 8-Br-cAMP. Control experiments with caged cGMP showed that flash-induced currents decayed within a few minutes because photoreleased cGMP was degraded by endogenous phosphodiesterase activity. The rise time of the 8-Br-cGMP-activated whole-cell current was consistent with a bimolecular reaction between channel and ligand.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Time-dependent current decline in cyclic GMP-gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes.

1. Amino acids with a charged or a polar residue in the putative pore region, between lysine 346 and glutamate 372 of the alpha-subunit of the cGMP-gated channel from bovine rods were mutated to a different amino acid. The mRNA encoding for the wild-type, i.e. the alpha-subunit, or mutant channels was injected in Xenopus laevis oocytes. 2. When glutamate 363 was mutated to asparagine, serine or alanine, the current activated by a steady cGMP concentration declined in mutant channels. No current decline was observed when glutamate 363 was mutated to aspartate, glutamine or glycine, when theronine 359, 360 and 364 were mutated to alanine or when other charged residues in the pore region were neutralized. 3. The amount of current decline and its time course were significantly voltage dependent. In mutant E363A the current decline developed within about 1.5 s at -100 mV, but in about 6 s at +100 mV. In the same mutant, the current declined to about 55% of its initial level at +100 mV and to about 10% at -100 mV. 4. The current decline in mutants E363A, E363S and E363N was only moderately dependent on the cGMP concentration (from 10 to 1000 microM) and was not caused by a reduced affinity of the mutant channels for cGMP. Analysis of current fluctuations at a single-channel level indicated that current decline was primarily caused by a decrease of the open probability. 5. The wild-type channel was not permeable to dimethylammonium. When glutamate 363 was replaced by a smaller residue such as serine, mutant channels became permeable to dimethylammonium. 6. The current decline observed in mutant channels is reminiscent of desensitization of ligand-gated channels and of inactivation of voltage-gated channels. These results suggest also that gating and permeation through the cGMP-gated channel from bovine rods are intrinsically coupled and that glutamate 363 is part of the molecular structure controlling both the gating and the narrowest region of the pore.

Amino Acid Sequence↗
PubMed1995

The multi-ion nature of the cGMP-gated channel from vertebrate rods.

1. Native cGMP-gated channels were studied in rod outer segments of the larval tiger salamander, Ambystoma tigrinum. The alpha-subunit of the cGMP-gated channel, here referred to as the wild type (WT), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside-out configuration and were activated by the addition of 100 or 500 microM cGMP. The current carried by monovalent cations was measured under voltage-clamp conditions. 2. In the presence of 110 mM Na+ in the extracellular medium and different amounts of Na+ in the intracellular medium, the I-V relations of the native channel could be described by a single-site model with a profile of Gibbs free energy with two barriers and a well. A similar result was obtained in the presence of 110 mM Li+ in the extracellular medium and different amounts of Li+ in the intracellular medium. The well depth was 1.4RT (where R is the gas constant and T is the absolute temperature) for both Li+ and Na+. 3. The I-V relations of the native channel in the presence of 110 mM Na+ on one side of the membrane and 110 mM Li+ on the other side could not be described by the same single-site model with identical values of barriers and well obtained in the presence of Li+ or Na+ alone: the well for Li+ had to be at least 4RT. 4. In the presence of mixtures of 110 mM Li+ and Cs+ on the cytoplasmic side of the membrane, an anomalous mole fraction effect was observed both in the native and the WT channel. No anomalous behaviour was seen in the presence of Li(+)-Na+ and Li(+)-NH4+ mixtures. 5. The anomalous mole fraction effect with mixtures of Li+ and Cs+ was not observed in the channel where glutamate 363 was mutated to a glutamine (E363Q) or an asparagine (E363N). When glutamate 363 was mutated to an aspartate (E363D), the anomalous mole fraction effect with mixtures of Li+ and Cs+ was still observed, although significantly reduced. 6. When lysine 346, arginine 369, aspartate 370 and glutamate 372 were neutralized by mutation to glutamine, the ion permeation through the mutant channels and the WT channel had largely similar properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗
PubMed1995

Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach.

Cyclic nucleotide-gated (CNG) cation channels contain two short sequence motifs--a residual voltage-sensor (S4) and a pore-forming (P) segment--that are reminiscent of similar segments in voltage-activated Shaker-type K+ channels. It has been tacitly assumed that CNG channels and this K+ channel subfamily share a common overall topology, characterized by a hydrophobic domain comprising six membrane-spanning segments. We have systematically investigated the topology of CNG channels from bovine rod photoreceptor and Drosophila melanogaster by a gene fusion approach using the bacterial reporter enzymes alkaline phosphatase and beta-galactosidase, which are active only in the periplasm and only in the cytoplasm, respectively. Enzymatic activity was determined after expression of fusion constructs in Escherichia coli. CNG channels were found to have six membrane-spanning segments, suggesting that CNG and Shaker-type K+ channels, albeit distant relatives within a gene superfamily of ion channels, share a common topology.

Alkaline Phosphatase↗
PubMed1995

Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels.

Sensory transduction in vertebrate photoreceptors and olfactory sensory neurons is mediated by cyclic nucleotide-gated (CNG) channels that conduct mono- and divalent cations. Ca2+ entering the cell through CNG channels intimately controls signaling pathways by regulating several key enzymes. Cloned CNG channels from photoreceptors and olfactory sensory neurons profoundly differ in their relative Ca2+ permeability, their blockage by external divalent cations, and the fraction of current carried by Ca2+. In particular, CNG channels from cone photoreceptors conduct significantly more Ca2+ than those from rod photoreceptors. Furthermore, the current through the olfactory CNG channel is entirely carried by Ca2+ at approximately 3 mM extracellular Ca2+. These results suggest that a major function of CNG channels is to provide a pathway for Ca2+ entry.

Animals↗
PubMed1995

Family of cyclic nucleotide gated ion channels.

A variety of different cyclic nucleotide gated ion channels have recently been identified using molecular cloning and electrophysiological techniques. Current research is focussed on the specific molecular determinants that endow these channels with their distinctive character of gating, selectivity and modulation. In some cases, it has been possible to identify the specific physiological roles of different cyclic nucleotide gated channels. Their interactions with Ca2+ and calmodulin are particularly important, and determine the specific functions these channels subserve in distinct cells.

Animals↗
PubMed1994

Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae.

Cyclic nucleotide-gated (CNG) ion channels serve as downstream targets of signalling pathways in vertebrate photoreceptors and olfactory sensory neurons. Whether CNG channels subserve similar functions in invertebrate photoreception and olfaction is unknown. We have cloned genomic DNA and cDNA encoding a cGMP-gated channel from Drosophila. The gene contains at least seven exons. Heterologous expression of cloned cDNA in both Xenopus oocytes and HEK 293 cells gives rise to functional ion channels. The Drosophila CNG channel is approximately 50-fold more sensitive to cGMP than to cAMP. The voltage dependence of blockage by divalent cations is different compared with the CNG channel of rod photoreceptors, and the Ca2+ permeability is much larger. The channel mRNA is expressed in antennae and the visual system of Drosophila. It is proposed that CNG channels are involved in transduction cascades of both invertebrate photoreceptors and olfactory sensillae.

Amino Acid Sequence↗
PubMed1994

Cloning and functional expression of a cyclic-nucleotide-gated channel from mammalian sperm.

Cyclic nucleotide-gated (CNG) channels serve as downstream targets of signalling pathways in vertebrate photoreceptor cells and olfactory sensory neurons (see ref. 1 for review). Ca2+ ions that enter through CNG channels intimately control these signalling pathways by regulating synthesis or hydrolysis of cyclic nucleotides, and by decreasing ligand sensitivity of CNG channels. Several lines of evidence suggest that cyclic nucleotides and Ca2+ play important roles in chemotaxis of invertebrate sperm and fertilization (see ref. 9 for review), whereas their mechanisms of action in vertebrate sperm are largely unknown. Here we report the cloning and functional expression of a novel CNG channel from bovine testis. The channel polypeptide was functionally localized in sperm, but is also specifically expressed in cone photoreceptor cells. These channels might be involved in chemotaxis of sperm by controlling Ca2+ entry through a cyclic-nucleotide signalling pathway.

Amino Acid Sequence↗
PubMed1994

A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity.

Ca2+ ions control the cGMP-gated channel of rod photoreceptor cells from the external and internal face. We studied ion selectivity and blockage by Ca2+ of wild-type and mutant channels in a heterologous expression system. External Ca2+ blocks the inward current at micromolar concentrations in a highly voltage-dependent manner. The blockage at negative membrane voltages shows a steep concentration dependence with a Hill coefficient of approximately 2. The blockage from the internal face requires approximately 1000-fold higher Ca2+ concentrations. Neutralization of a glutamate residue (E363) in the putative pore region between transmembrane segments H4 and H5 induces outward rectification and changes relative ion conductances but leaves relative ion permeabilities nearly unaffected. The current blockage at -80 mV requires approximately 2000-fold higher external Ca2+ concentrations and the voltage dependence is almost abolished. These results demonstrate that E363 represents a binding site for monovalent and divalent cations and resides in the pore lumen.

Amino Acid Sequence↗