Reinhard Seifert

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 (see ref. 1 for a review). 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 thesuperfamily of voltage-gated and cyclic-nucleotide-gated channels.

The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm

In the oviduct, cumulus cells that surround the oocyte release progesterone. In human sperm, progesterone stimulates a Ca2+ increase by a non-genomic mechanism. The Ca2+ signal has been proposed to control chemotaxis, hyperactivation and acrosomal exocytosis of sperm. However, the underlying signalling mechanism has remained mysterious. Here we show that progesterone activates the sperm-specific, pH-sensitive CatSper Ca2+ channel. We found that both progesterone and alkaline pH stimulate a rapid Ca2+ influx with almost no latency, incompatible with a signalling pathway involving metabotropic receptors and second messengers. The Ca2+ signals evoked by alkaline pH and progesterone are inhibited by the Cav channel blockers NNC 55-0396 and mibefradil. Patch-clamp recordings from sperm reveal an alkaline-activated current carried by mono- and divalent ions that exhibits all the hallmarks of sperm-specific CatSper Ca2+ channels. Progesterone substantially enhances the CatSper current. The alkaline- and progesterone-activated CatSper current is inhibited by both drugs. Our results resolve a long-standing controversy over the non-genomic progesterone signalling. In human sperm, either the CatSper channel itself or an associated protein serves as the non-genomic progesterone receptor. The identification of CatSper channel blockers will greatly facilitate the study of Ca2+ signalling in sperm and help to define further the physiological role of progesterone and CatSper.

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.

Hyperpolarization-activated channels HCN1 and HCN4 mediate responses to sour stimuli

Sour taste is initiated by protons acting at receptor proteins or channels. In vertebrates, transduction of this taste quality involves several parallel pathways. Here we examine the effects of sour stimuli on taste cells in slices of vallate papilla from rat. From a subset of cells, we identified a hyperpolarization-activated current that was enhanced by sour stimulation at the taste pore. This current resembled Ih found in neurons and cardio-myocytes, a current carried by members of the family of hyperpolarization-activated and cyclic-nucleotide-gated (HCN) channels. We show by in situ hybridization and immunohistochemistry that HCN1 and HCN4 are expressed in a subset of taste cells. By contrast, gustducin, the G-protein involved in bitter and sweet taste, is not expressed in these cells. Lowering extracellular pH causes a dose-dependent flattening of the activation curve of HCN channels and a shift in the voltage of half-maximal activation to more positive voltages. Our results indicate that HCN channels are gated by extracellular protons and may act as receptors for sour taste.

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.

Fast manipulation of cellular cAMP level by light in vivo

The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACα and PACβ. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.

Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates.

Cyclic nucleotide‐gated (CNG) channels play an important role in Ca2+ signaling in many cells. CNG channels from various tissues differ profoundly in their Ca2+ permeation properties. Using the voltage‐dependent Ca2+ blockage of monovalent current in wild‐type channels, chimeric constructs and point mutants, we have identified structural elements that determine the distinctively different interaction of Ca2+ with CNG channels from rod and cone photoreceptors and olfactory neurons. Segments S5 and S6 and the extracellular linkers flanking the pore region are the only structural elements that account for the differences between channels. Ca2+ blockage is strongly modulated by external pH. The different pH dependence of blockage suggests that the pKa of intrapore glutamates and their protonation pattern differ among channels. The results support the hypothesis that the S5–pore–S6 module, by providing a characteristic electrostatic environment, determines the protonation state of pore glutamates and thereby controls Ca2+ affinity and permeation in each channel type.

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.

Cooperative and uncooperative cyclic-nucleotide-gated ion channels

Ion channels gated by cyclic nucleotides serve multiple functions in sensory signaling in diverse cell types ranging from neurons to sperm. Newly discovered members from bacteria and marine invertebrates provide a wealth of structural and functional information on this channel family. A hallmark of classical tetrameric cyclic-nucleotide-gated channels is their cooperative activation by binding of several ligands. By contrast, the new members seem to be uncooperative, and binding of a single ligand molecule suffices to open these channels. These new findings provide a fresh look at the mechanism of allosteric activation of ion channels.

Pacemaker oscillations in heart and brain: a key role for hyperpolarization-activated cation channels.

Rhythmic activity of single cells or multicellular networks is a common feature of all organisms. The oscillatory activity is characterized by time intervals of several seconds up to many hours. Cellular rhythms govern the beating of the heart, the swimming behavior of sperm, cycles of sleep and wakefulness, breathing, and the release of hormones. Many neurons in the brain and cardiac cells are characterized by endogenous rhythmic activity, which relies on a complex interplay between several distinct ion channels. In particular, one type of ion channel plays a prominent role in the control of rhythmic electrical activity since it determines the frequency of the oscillations. The activity of the channels is thus setting the “pace” of the oscillations; therefore, these channels are often referred to as “pacemaker” channels. Despite their obvious important physiological function, it was not until recently that genes encoding pacemaker channels have been identified. Because both hyperpolarization and cyclic nucleotides are key elements that control their activity, pacemaker channels have now been designated hyperpolarization-activated and cyclic nucleotide–gated (HCN) channels. The molecular identification of the channels and the upcoming studies on their properties in heterologous systems will certainly enhance our understanding of “pacemaking” in physiological systems. This review gives a brief insight into the physiological importance of these channels and sums up what we have learned since the first cloning of genes succeeded (for recent reviews, see also Clapham 1998; Luüthi and McCormick 1998a; Biel et al. 1999; Ludwig, Zong, Hofmann, et al. 1999; Santoro and Tibbs 1999). (Chronobiology International, 17(4), 453–469, 2000)