Wolfgang Bönigk

Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels

Signal transduction in vertebrate rod and cone photoreceptor cells involves ion channels that are directly gated by the internal messenger cGMP. Rods and each type of cones express genetically related yet different forms of photopigments. Enzymes that control the light-stimulated hydrolysis of cGMP in rods and cones are also the product of distinct genes. Two different cDNA clones encoding cGMP-gated channels have been characterized from the chicken retina. Expression of cDNAs in Xenopus oocytes gives rise to cGMP-stimulated channel activity. Antibodies against a synthetic peptide specific for the C-terminal amino acid sequence derived from one clone stain outer segments of cone but not rod photoreceptors. Therefore chicken rod and cone cells each express different forms of cGMP-gated channels that are genetically related to each other. Expression in COS-1 cells produces the complete form of both channel polypeptides, whereas Western blot analysis indicates that channels in outer segment membranes are present in a processed form that is significantly shorter than the full-length polypeptide.

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+.

An Atypical CNG Channel Activated by a Single cGMP Molecule Controls Sperm Chemotaxis

The ability of a single molecule of cGMP to activate the K+-selective cyclic nucleotide–gated channel allows sea urchin sperm to find an egg. Finding an Egg in an Ocean Sperm of the sea urchin Arbacia punctulata, which are released into the ocean and must find their way to an egg before fertilization can take place, can sense and respond to a single molecule of the egg-derived chemoattractant resact. This response depends on the production of guanosine 3′,5′-monophosphate (cGMP) and the consequent activation of K+-selective cyclic nucleotide–gated (CNGK) channels, which leads to production of an intracellular calcium signal that regulates movement of the sperm flagellum and thereby the direction in which the sperm cell swims. After cloning the A. punctulata CNGK, Bönigk et al. combined mutational analysis with optical analysis and electrophysiology to explore the mechanisms responsible for this sensitivity. They found that, although CNGK contains four repeating regions, each of which resembles a cyclic nucleotide–gated (CNG) channel subunit and contains a cyclic nucleotide–binding domain, it is activated through binding of only a single molecule of cGMP. Using a compound that cages cGMP and becomes fluorescent after its release, they were able to calibrate the system and determine that fewer than 50 molecules of cGMP were required to mediate the Ca2+ response to a single molecule of resact. Sperm of the sea urchin Arbacia punctulata can respond to a single molecule of chemoattractant released by an egg. The mechanism underlying this extreme sensitivity is unknown. Crucial signaling events in the response of A. punctulata sperm to chemoattractant include the rapid synthesis of the intracellular messenger guanosine 3′,5′-monophosphate (cGMP) and the ensuing membrane hyperpolarization that results from the opening of potassium–selective cyclic nucleotide–gated (CNGK) channels. Here, we use calibrated photolysis of caged cGMP to show that ~45 cGMP molecules are generated during the response to a single molecule of chemoattractant. The CNGK channel can respond to such small cGMP changes because it is exquisitely sensitive to cGMP and activated in a noncooperative fashion. Like voltage-activated Cav and Nav channels, the CNGK polypeptide consists of four homologous repeat sequences. Disabling each of the four cyclic nucleotide–binding sites through mutagenesis revealed that binding of a single cGMP molecule to repeat 3 is necessary and sufficient to activate the CNGK channel. Thus, CNGK has developed a mechanism of activation that is different from the activation of other CNG channels, which requires the cooperative binding of several ligands and operates in the micromolar rather than the nanomolar range.

Subunits act independently in a cyclic nucleotide‐activated K+ channel

Ion channels gated by cyclic nucleotides have crucial roles in neuronal excitability and signal transduction of sensory neurons. Here, we studied ligand binding of a cyclic nucleotide‐activated K+ channel from Mesorhizobium loti and its isolated cyclic nucleotide‐binding domain. The channel and the binding domain alone bind cyclic AMP with similar affinity in a non‐cooperative manner. The cAMP sensitivities of binding and activation coincide. Thus, each subunit in the tetrameric channel acts independently of the others. The binding and gating properties of the bacterial channel are distinctively different from those of eukaryotic cyclic nucleotide‐gated channels.

The Ca2+-activated K+ current of human sperm is mediated by Slo3

Sperm are equipped with a unique set of ion channels that orchestrate fertilization. In mouse sperm, the principal K+ current (IKSper) is carried by the Slo3 channel, which sets the membrane potential (Vm) in a strongly pHi-dependent manner. Here, we show that IKSper in human sperm is activated weakly by pHi and more strongly by Ca2+. Correspondingly, Vm is strongly regulated by Ca2+ and less so by pHi. We find that inhibitors of Slo3 suppress human IKSper, and we identify the Slo3 protein in the flagellum of human sperm. Moreover, heterologously expressed human Slo3, but not mouse Slo3, is activated by Ca2+ rather than by alkaline pHi; current–voltage relations of human Slo3 and human IKSper are similar. We conclude that Slo3 represents the principal K+ channel in human sperm that carries the Ca2+-activated IKSper current. We propose that, in human sperm, the progesterone-evoked Ca2+ influx carried by voltage-gated CatSper channels is limited by Ca2+-controlled hyperpolarization via Slo3. DOI: http://dx.doi.org/10.7554/eLife.01438.001

Corticotropin-Releasing Factor (CRF) Agonists Stimulate Testosterone Production in Mouse Leydig Cells through CRF Receptor-1

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nm for hCRF, antagonized by the CRF antagonist α-helical CRF9–41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to[ 11-d-Thr,12-d-Phe]- and[ 13-d-His,14-d-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smalle...

The CatSper channel controls chemosensation in sea urchin sperm

Sperm guidance is controlled by chemical and physical cues. In many species, Ca2+ bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca2+ bursts. The underlying Ca2+ channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca2+ channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant‐evoked Ca2+ influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca2+ bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization.

Structural Features of Cyclic Nucleotide-Gated Channels

Cyclic nucleotide-gated (CNG) cation channels represent a novel class of ion channels that are directly and cooperatively gated by the binding of guanosine 3’,5’-cyclic monophosphate (cGMP) or adenosi

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

The sea urchin sperm guanylyl cyclase chemoreceptor achieves ultrasensitive signal detection and precise signal modulation through high receptor density, subnanomolar ligand affinity, and sequential dephosphorylation.

Differential Regulation by Cyclic Nucleotides of the CNGA4 and CNGB1b Subunits in Olfactory Cyclic Nucleotide-Gated Channels

Different subunits of olfactory CNG channels enable specific regulation of channel activity by cAMP or cGMP. Unraveling Regulation of Olfactory CNG Channels by Cyclic Nucleotides Heterotetrameric olfactory cyclic nucleotide–gated (CNG) channels undergo complex regulation by cyclic nucleotides cAMP and cGMP, with activation in response to binding to the CNGA2 subunit well established. Nache et al. used a fluorescent cGMP (fcGMP) analog and confocal patch-clamp fluorometry, which allows simultaneous analysis of ligand binding and channel activity, to explore channel regulation in response to ligand binding. Analysis of heterotetrameric channels containing various combinations of the CNGA4, CNGB1b, and CNGA2 subunits, with and without mutations that compromised cyclic nucleotide binding, showed that both the CNGA4 and the CNGA2 subunits, but not the CNGB1b subunit, bound and activated the channel in response to fcGMP. In contrast, all three subunits contributed to channel activation by cAMP. Thus, the presence of the CNGB1b subunit may enable the channel to discriminate between cAMP and cGMP. Olfactory cyclic nucleotide–gated (CNG) ion channels are essential contributors to signal transduction of olfactory sensory neurons. The activity of the channels is controlled by the cyclic nucleotides guanosine 3′,5′-monophosphate (cGMP) and adenosine 3′,5′-monophosphate (cAMP). The olfactory CNG channels are composed of two CNGA2 subunits, one CNGA4 and one CNGB1b subunit, each containing a cyclic nucleotide–binding domain. Using patch-clamp fluorometry, we measured ligand binding and channel activation simultaneously and showed that cGMP activated olfactory CNG channels not only by binding to the two CNGA2 subunits but also by binding to the CNGA4 subunit. In a channel in which the CNGA2 subunits were compromised for ligand binding, cGMP binding to CNGA4 was sufficient to partly activate the channel. In contrast, in heterotetrameric channels, the CNGB1b subunit did not bind cGMP, but channels with this subunit showed activation by cAMP. Thus, the modulatory subunits participate actively in translating ligand binding to activation of heterotetrameric olfactory CNG channels and enable the channels to differentiate between cyclic nucleotides.