Biomedical subjects

S Frings

Publications and source records attributed to S Frings.

30 records · Page 2Linked to original sources

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↗

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↗

PubMed1993

Protein kinase C sensitizes olfactory adenylate cyclase.

Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory sensitivity.

Adenylyl Cyclases↗

PubMed1992

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.

Animals↗

PubMed1991

Current recording from sensory cilia of olfactory receptor cells in situ. I. The neuronal response to cyclic nucleotides.

The olfactory mucosa of the frog was isolated, folded (the outer, ciliated side faced outward), and separately superfused with Ringers solution on each side. A small number of sensory cilia (one to three) were pulled into the orifice of a patch pipette and current was recorded from them. Fast bipolar current transients, indicating the generation of action potentials by the receptor cells, were transmitted to the pipette, mainly through the ciliary capacitance. Basal activity was near 1.5 spikes s-1. Exposure of apical membrane areas outside of the pipette to permeant analogues of cyclic nucleotides, to forskolin, and to phosphodiesterase inhibitors resulted in a dose-dependent acceleration of spike rate of all cells investigated. Values of 10-20 s-1 were reached. These findings lend further support to the notion that cyclic nucleotides act as second messengers, which cause graded membrane depolarization and thereby a graded increase in spike rate. The stationary spike rate induced by forskolin was very regular, while phosphodiesterase inhibitors caused (in the same cell) an irregular pattern of bursts of spikes. The response of spike rate was phasic-tonic in the case of strong stimulation, even when elicited by inhibitors of phosphodiesterase or by analogues of cyclic nucleotides that are not broken down by the enzyme. Thus, one of the mechanisms contributing to desensitization appears to operate at the level of the nucleotide-induced ciliary conductance. However, desensitization at this level was slow and only partial, in contrast to results obtained with isolated, voltage-clamped receptor cells.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Current recording from sensory cilia of olfactory receptor cells in situ. II. Role of mucosal Na+, K+, and Ca2+ ions.

Action potential-driven current transients were recorded from sensory cilia and used to monitor the spike frequency generated by olfactory receptor neurons, which were maintained in their natural position in the sensory epithelium. Both basal and messenger-induced activities, as elicited with forskolin or cyclic nucleotides, were dependent on the presence of mucosal Na+. The spike rate decreased to approximately 20% when mucosal Na+ was lowered from 120 to 60 mM (replaced by N-methyl-D-glucamine+), without clear changes in amplitude and duration of the recorded action potential-driven transients. Mucosal Ca2+ and Mg2+ blocked spike discharge completely when increased from 1 to 10 mM in Ringer solution. Lowering mucosal Ca2+ below 1 mM increased the spike rate. These results can be explained by the presence of a cyclic nucleotide-dependent, Ca(2+)-sensitive cation conductance, which allows a depolarizing Na+ inward current to flow through the apical membrane of in situ receptor cells. A conductance with these properties, thought to provide the receptor current, was first described for isolated olfactory cells by Nakamura and Gold (1987. Nature (Lond.). 325:442-444). The forskolin-stimulated spike rate decreased when l-cis-diltiazem, a known blocker of the cyclic nucleotide-dependent receptor current, was added to the mucosal solution. Spike rate also decreased when the mucosal K+ concentration was lowered. Mucosal Ba2+ and 4-aminopyridine, presumably by means of cell depolarization, rapidly increased the spike rate. This suggests the presence of apical K+ channels that render the receptor cells sensitive to the K+ concentration of the olfactory mucus. With a slower time course, mucosal Ba2+ and 4-aminopyridine decreased the amplitude and caused rectification of the fast current transients (prolongation of action potentials). Abolishment of the apical Na+ current (by removal of mucosal Na+), as indicated by a strong decrease in spike rate, could be counteracted by adding 10 mM Ba2+ or 1 mM 4-aminopyridine to the mucosal solution, which re-established spiking. Similarly, blockage of the apical cation conductance with 10 mM Ca could be counteracted by adding 10 mM Ba2+ or by raising the mucosal K+ concentration. Thus mucosal concentrations of Na+, K+, and Ca2+ will jointly affect the sensitivity of odor detection.

Animals↗

PubMed1990

Single unit recording from olfactory cilia.

Sensory cilia from olfactory receptor cells can be pulled into a patch pipette located above the mucus layer of an olfactory mucosa. While the pipette does not form a tight electrical seal with the ciliary membrane, it nevertheless allows to record current transients driven by action potentials arising in the olfactory neuron. This method is an alternative to single-unit-recording with electrodes pushed into the mucosa and, in some respects, to patch clamp recordings from isolated olfactory cells. Its advantage is technical simplicity and minimal disturbance of the neuron from which signals are derived. Less than 5% of the chemosensitive apical surface of the neuron is covered by the pipette. The neuron remains in situ and its cilia remain covered with some mucus. (However, mucus is in part dissolved by the bathing solution). Odorant thresholds in the picomolar range were thus obtained.

Action Potentials↗

PubMed1988

Odorant response of isolated olfactory receptor cells is blocked by amiloride.

Olfactory receptor cells were isolated from the nasal mucosa of Rana esculenta and patch clamped. Best results were obtained with free-floating cells showing ciliary movement. 1) On-cell mode: Current records were obtained for up to 50 min. Under control conditions they showed only occasional action potentials. The odorants cineole, amyl acetate and isobutyl methoxypyrazine were applied in saline by prolonged superfusion. At 500 nanomolar they elicited periodic bursts of current transients arising from cellular action potentials. The response was rapidly, fully and reversibly blocked by 50 microM amiloride added to the odorant solution. With 10 microM amiloride, the response to odorants was only partially abolished. 2) Whole-cell mode: Following breakage of the patch, the odorant response was lost within 5 to 15 min. Prior to this, odorants evoked a series of slow transient depolarizations (0.1/sec, 45 mV peak to peak) which reached threshold and thus elicited the periodic discharge of action potentials. These slow depolarizing waves were reversibly blocked by amiloride, which stabilized the membrane voltage between -80 and -90 mV. We conclude that amiloride inhibits chemosensory transduction of olfactory receptor cells, probably by blocking inward current pathways which open in response to odorants.

Amiloride↗

PubMed1988

Single-channel recordings from the apical membrane of the toad urinary bladder epithelial cell.

The patch-clamp technique for the recording of single-channel currents was used to investigate the activity of ion channels in the intact epithelium of the toad urinary bladder. High resistance seals were obtained from the apical membrane of tightly stretched tissue. Single-channel recordings revealed the activity of a variety of ion channels that could be classified in 4 groups according to their mean ion conductances, ranging from 5 to 59 pS. In particular, we observed highly selective, amiloride-sensitive Na channels with a mean conductance of 4.8 pS, channels with a similar conductance that were not Na-selective and channels with mean conductance values of 17-58 pS that were mostly seen after stimulation of the tissue with vasopressin or cAMP. When inside-out patches from the apical membrane were exposed to 110 mM fluoride, large conductances (86-490 pS) appeared.

Amiloride↗

PubMedDate not supplied

Synthesis, photochemistry and application of (7-methoxycoumarin-4-yl)methyl-caged 8-bromoadenosine cyclic 3',5'-monophosphate and 8-bromoguanosine cyclic 3',5'-monophosphate photolyzed in the nanosecond time region.

New caged derivatives of hydrolysis-resistant 8-bromoadenosine cyclic 3',5'-monophosphate (8-Br-cAMP) and 8-bromoguanosine cyclic 3',5'-monophosphate (8-Br-cGMP) are described. The compounds are the axial and equatorial isomers of the (7-methoxycoumarin-4-yl)methyl (MCM) esters of cyclic nucleotides. Synthesis is accomplished by treatment of 4-bromomethyl-7-methoxycoumarin with the tetra-n-butylammonium salts of the 8-bromo-substituted cyclic nucleotides or with the free acids of 8-Br-cAMP and 8-Br-cGMP in the presence of silver(I) oxide. MCM-caged 8-Br-cAMP and MCM-caged 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light within a few nanoseconds. They show favorable absorption properties and quantum yields and are resistant to hydrolysis in aqueous buffer solutions. The moderate fluorescence properties of the caged compounds in comparison with the strongly fluorescent 4-hydroxymethyl-7-methoxycoumarin (MCM-OH) photoproduct allow the indirect estimation of the amount of photolytically released cyclic nucleotides in aqueous buffer solutions using fluorescence measurements. Their usefulness for physiological studies has been examined in a mammalian cell line expressing the cyclic nucleotide-gated ion channel of bovine olfactory sensory neurons using the patch-clamp technique and confocal laser scanning microscopy. The caged compounds serve as efficient and rapid intracellular sources of 8-Br-cAMP and 8-Br-cGMP. However, at least in HEK 293 cells, fluorescence signals cannot be used to monitor the photolysis of MCM-caged 8-Br-cAMP and 8-Br-cGMP, due to quenching of the fluorescence of MCM-OH.

Animals↗

PubMedDate not supplied

[Cobra venom contains a protein belongs to the CRISP family].

Amino acid sequences of several fragments of the 25 k protein (molecular mass 24,953 Da) previously isolated from cobra Naja kaouthia (Kukhtina et al. Bioorg. Khim., 2000, vol. 26, pp. 803-807) were determined. Their comparison with the primary structures of known proteins showed that the 25 k protein belongs to the CRISP family and is the first protein of this type identified in cobra venoms.

Amino Acid Sequence↗