Stephan Frings

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.

Neuronal Ca2+ -activated Cl- channels--homing in on an elusive channel species.

Ca2+ -activated Cl- channels control electrical excitability in various peripheral and central populations of neurons. Ca2+ influx through voltage-gated or ligand-operated channels, as well as Ca2+ release from intracellular stores, have been shown to induce substantial Cl- conductances that determine the response to synaptic input, spike rate, and the receptor current of various kinds of neurons. In some neurons, Ca2+ -activated Cl- channels are localized in the dendritic membrane, and their contribution to signal processing depends on the local Cl- equilibrium potential which may differ considerably from those at the membranes of somata and axons. In olfactory sensory neurons, the channels are expressed in ciliary processes of dendritic endings where they serve to amplify the odor-induced receptor current. Recent biophysical studies of signal transduction in olfactory sensory neurons have yielded some insight into the functional properties of Ca2+ -activated Cl- channels expressed in the chemosensory membrane of these cells. Ion selectivity, channel conductance, and Ca2+ sensitivity have been investigated, and the role of the channels in the generation of receptor currents is well understood. However, further investigation of neuronal Ca2+ -activated Cl- channels will require information about the molecular structure of the channel protein, the regulation of channel activity by cellular signaling pathways, as well as the distribution of channels in different compartments of the neuron. To understand the physiological role of these channels it is also important to know the Cl- equilibrium potential in cells or in distinct cell compartments that express Ca2+ -activated Cl- channels. The state of knowledge about most of these aspects is considerably more advanced in non-neuronal cells, in particular in epithelia and smooth muscle. This review, therefore, collects results both from neuronal and from non-neuronal cells with the intent of facilitating research into Ca2+ -activated Cl- channels and their physiological functions in neurons.

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.

Chloride Accumulation in Mammalian Olfactory Sensory Neurons

The generation of an excitatory receptor current in mammalian olfactory sensory neurons (OSNs) involves the sequential activation of two distinct types of ion channels: cAMP-gated Ca2+-permeable cation channels and Ca2+-gated Cl- channels, which conduct a depolarizing Cl- efflux. This unusual transduction mechanism requires an outward-directed driving force for Cl-, established by active accumulation of Cl- within the lumen of the sensory cilia. We used two-photon fluorescence lifetime imaging microscopy of the Cl--sensitive dye 6-methoxy-quinolyl acetoethyl ester to measure the intracellular Cl- concentration in dendritic knobs of OSNs from mice and rats. We found a uniform intracellular Cl- concentration in the range of 40-50 mm, which is indicative of active Cl- accumulation. Functional assays and PCR experiments revealed that NKCC1-mediated Cl- uptake through the apical membrane counteracts Cl- depletion in the sensory cilia, and thus maintains the responsiveness of OSNs to odor stimulation. To permit Cl- accumulation, OSNs avoid the “chloride switch”: they do not express KCC2, the main Cl- extrusion cotransporter operating in neurons of the adult CNS. Cl- accumulation provides OSNs with the driving force for the depolarizing Cl- current that is the basis of the low-noise receptor current in these neurons.

Ca2+ permeation in cyclic nucleotide‐gated channels

Cyclic nucleotide‐gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non‐excitable cells, co‐assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+‐binding affinity of the intrapore cation‐binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.

Das vegetative Nervensystem

Im zentralen Hohlengrau des dritten Ventrikels (siehe Abb. 121 u. 157), liegen eine Reihe von Ganglienzellengruppen, welche offenbar der Regulation wichtiger Lebensvorgange dienen: Die Aufrechterhaltung der Korpertemperatur auf ihrer normalen Hohe wird dadurch bewerkstelligt, das bei Abkuhlung eine vermehrte Warmeproduktion und das bei Erhohung der Korpertemperatur eine vermehrte Warmeabgabe durch Erweiterung der peripherischen Blutgefase und durch Schweisproduktion vom Zentrum aus angeregt wird. Ferner geschieht die Regulation der Wasserausscheidung durch den Harn und den Schweis unter dem Einflus dieser grauen Substanz und des damit zusammenhangenden Hinterlappens der Hypophyse, bei deren Erkrankung eine ubermasige Wasserausscheidung durch den Harn zustande kommt. Spritzt man dagegen den Saft einer normalen Hypophyse subcutan ein, so vermindert sich die Harnsekretion. Durch die zentrale Regulation der Wasserausscheidung wird erreicht, das der Wassergehalt des Blutes und der Gewebe auf normaler Hohe bleibt. Im Zusammenhang damit wird auch der Kochsalzgehalt des Blutes und der Gewebssafte in dem osmotischen Bereich einer physiologischen Kochsalzlosung gehalten. Sinkt der Wassergehalt des Blutes und der Gewebe unter ein gewisses Mas, so treten Osophaguskontraktionen auf, welche das Durstgefuhl zum Ausdruck bringen.

The Ca-activated Cl channel and its control in rat olfactory receptor neurons

Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP-signaling cascade, which results in the opening of nonselective, cyclic nucleotide–gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here some general properties of this Ca-activated Cl channel in rat, as well as its functional interplay with the CNG channel, by using inside-out membrane patches excised from ORN dendritic knobs/cilia. At physiological concentrations of external divalent cations, the maximally activated Cl current was ∼30 times as large as the CNG current. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels opened by cAMP. The magnitude of the Cl current depended on the strength of Ca buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were ∼8 and 62 μm−2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current.

A family of octapamine receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogaster

In invertebrates, the biogenic‐amine octopamine is an important physiological regulator. It controls and modulates neuronal development, circadian rhythm, locomotion, ‘fight or flight’ responses, as well as learning and memory. Octopamine mediates its effects by activation of different GTP‐binding protein (G protein)‐coupled receptor types, which induce either cAMP production or Ca2+ release. Here we describe the functional characterization of two genes from Drosophila melanogaster that encode three octopamine receptors. The first gene (Dmoa1) codes for two polypeptides that are generated by alternative splicing. When heterologously expressed, both receptors cause oscillatory increases of the intracellular Ca2+ concentration in response to applying nanomolar concentrations of octopamine. The second gene (Dmoa2) codes for a receptor that specifically activates adenylate cyclase and causes a rise of intracellular cAMP with an EC50 of ∼3 × 10−8 m octopamine. Tyramine, the precursor of octopamine biosynthesis, activates all three receptors at ≥ 100‐fold higher concentrations, whereas dopamine and serotonin are non‐effective. Developmental expression of Dmoa genes was assessed by RT–PCR. Overlapping but not identical expression patterns were observed for the individual transcripts. The genes characterized in this report encode unique receptors that display signature properties of native octopamine receptors.

Calmodulin permanently associates with rat olfactory CNG channels under native conditions

An important mechanism by which vertebrate olfactory sensory neurons rapidly adapt to odorants is feedback modulation of the Ca2+-permeable cyclic nucleotide–gated (CNG) transduction channels. Extensive heterologous studies of homomeric CNGA2 channels have led to a molecular model of channel modulation based on the binding of calcium-calmodulin to a site on the cytoplasmic amino terminus of CNGA2. Native rat olfactory CNG channels, however, are heteromeric complexes of three homologous but distinct subunits. Notably, in heteromeric channels, we found no role for CNGA2 in feedback modulation. Instead, an IQ-type calmodulin-binding site on CNGB1b and a similar but previously unidentified site on CNGA4 are necessary and sufficient. These sites seem to confer binding of Ca2+-free calmodulin (apocalmodulin), which is then poised to trigger inhibition of native channels in the presence of Ca2+.

Regulation of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are found in several cell types, and are best studied in photoreceptors and olfactory sensory neurons. There, CNG channels are gated by the second messengers of the visual and olfactory signalling cascades, cGMP and cAMP respectively, and operate as transduction channels generating the stimulus-induced receptor potentials. In visual and olfactory sensory cells CNG channels conduct cationic currents. Calcium can contribute a large fraction of this current, and calcium influx serves a modulatory role in CNG-channel mediated signal transduction. There have been recent developments in our understanding of how the regulation of CNG channels contributes to the physiological properties of photoreceptors and olfactory sensory cells, and in particular on the role of calcium-mediated feedback.