Membrane-bound Guanylyl Cyclase Research Articles

An engineered membrane-bound guanylyl cyclase with light-switchable activity

BackgroundMicrobial rhodopsins vary in their chemical properties, from light sensitive ion transport to different enzymatic activities. Recently, a novel family of two-component Cyclase (rhod)opsins (2c-Cyclop) from the green algae Chlamydomonas reinhardtii and Volvox carteri was characterized, revealing a light-inhibited guanylyl cyclase (GC) activity. More genes similar to 2c-Cyclop exist in algal genomes, but their molecular and physiological functions remained uncharacterized.ResultsChlamyopsin-5 (Cop5) from C. reinhardtii is related to Cr2c-Cyclop1 (Cop6) and can be expressed in Xenopus laevis oocytes, but shows no GC activity. Here, we exchanged parts of Cop5 with the corresponding ones of Cr2c-Cyclop1. When exchanging the opsin part of Cr2c-Cyclop1 with that of Cop5, we obtained a bi-stable guanylyl cyclase (switch-Cyclop1) whose activity can be switched by short light flashes. The GC activity of switch-Cyclop1 is increased for hours by a short 380 nm illumination and switched off (20-fold decreased) by blue or green light. switch-Cyclop1 is very light-sensitive and can half-maximally be activated by ~ 150 photons/nm2 of 380 nm (~ 73 J/m2) or inhibited by ~ 40 photons/nm2 of 473 nm (~ 18 J/m2).ConclusionsThis engineered guanylyl cyclase is the first light-switchable enzyme for cGMP level regulation. Light-regulated cGMP production with high light-sensitivity is a promising technique for the non-invasive investigation of the effects of cGMP signaling in many different tissues.

Cardiac natriuretic peptides.

Investigations into the mixed muscle-secretory phenotype of cardiomyocytes from the atrial appendages of the heart led to the discovery that these cells produce, in a regulated manner, two polypeptide hormones - the natriuretic peptides - referred to as atrial natriuretic factor or atrial natriuretic peptide (ANP) and brain or B-type natriuretic peptide (BNP), thereby demonstrating an endocrine function for the heart. Studies on the gene encoding ANP (NPPA) initiated the field of modern research into gene regulation in the cardiovascular system. Additionally, ANP and BNP were found to be the natural ligands for cell membrane-bound guanylyl cyclase receptors that mediate the effects of natriuretic peptides through the generation of intracellular cGMP, which interacts with specific enzymes and ion channels. Natriuretic peptides have many physiological actions and participate in numerous pathophysiological processes. Important clinical entities associated with natriuretic peptide research include heart failure, obesity and systemic hypertension. Plasma levels of natriuretic peptides have proven to be powerful diagnostic and prognostic biomarkers of heart disease. Development of pharmacological agents that are based on natriuretic peptides is an area of active research, with vast potential benefits for the treatment of cardiovascular disease.

Crustacean hyperglycemic hormone (CHH) affects hemocyte intracellular signaling pathways to regulate exocytosis and immune response in white shrimp Litopenaeus vannamei

Recombinant Litopenaeus vannamei CHH (rLvCHH) was obtained from a bacterial expression system and the intracellular signaling pathways involved in exocytosis and immune response after rLvCHH injection (0.2 and 2 μg/shrimp) was investigated in this study. The results showed that CHH contents increased 51.4%–110.2% (0.2 μg/shrimp) and 65.0%–211.3% (2 μg/shrimp) of the control level. And the contents of three biogenic amines in hemolymph presented a similar variation pattern after rLvCHH injection, but reached the highest level at different time points. Furthermore, the mRNA expression levels of membrane-bound guanylyl cyclase (mGC) (1.20–1.93 fold) and biogenic amine receptors, including type 2 dopamine receptor (DA2R) (0.72-0.89 fold), α2 adrenergic receptor (α2-AR) (0.72-0.91 fold) and 5-HT7 receptor (5-HT7R) (1.37–3.49 fold) in hemocytes were changed consistently with their ligands. In addition, the second messenger and protein kinases shared a similar trend and reached the maximum at the same time respectively. The expression levels of nuclear transcription factor (cAMP response element-binding protein, CREB) and exocytosis-related proteins transcripts were basically overexpressed after rLvCHH stimulation, which reached the peaks at 1 h or 3 h. Eventually, the phenoloxidase (PO) activity (37.4%–158.5%) and antibacterial activity (31.8%–122.3%) in hemolymph were dramatically enhanced within 6 h, while the proPO activity in hemocytes significantly decreased (11.2%–62.6%). Collectively, these results indicate that shrimps L. vannamei could carry out a simple but ‘smart’ NEI regulation by releasing different neuroendocrine factors at different stages after rLvCHH stimulation, which could couple with their receptors and trigger the downstream signaling pathways during the immune responses in hemocytes.

Sp1 mediates phorbol ester (PMA)-induced expression of membrane-bound guanylyl cyclase GC-A in human monocytic THP-1 cells.

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.

Cardiac natriuretic peptides promote adipose ‘browning’ through mTOR complex-1

ObjectiveActivation of thermogenesis in brown adipose tissue (BAT) and the ability to increase uncoupling protein 1 (UCP1) levels and mitochondrial biogenesis in white fat (termed ‘browning’), has great therapeutic potential to treat obesity and its comorbidities because of the net increase in energy expenditure. β-adrenergic-cAMP-PKA signaling has long been known to regulate these processes. Recently PKA-dependent activation of mammalian target of rapamycin complex 1 (mTORC1) was shown to be necessary for adipose ‘browning’ as well as proper development of the interscapular BAT. In addition to cAMP-PKA signaling pathways, cGMP-PKG signaling also promotes this browning process; however, it is unclear whether or not mTORC1 is also necessary for cGMP-PKG induced browning. MethodActivation of mTORC1 by natriuretic peptides (NP), which bind to and activate the membrane-bound guanylyl cyclase, NP receptor A (NPRA), was assessed in mouse and human adipocytes in vitro and mouse adipose tissue in vivo. ResultsActivation of mTORC1 by NP-cGMP signaling was observed in both mouse and human adipocytes. We show that NP-NPRA-PKG signaling activate mTORC1 by direct PKG phosphorylation of Raptor at Serine 791. Administration of B-type natriuretic peptide (BNP) to mice induced Ucp1 expression in inguinal adipose tissue in vivo, which was completely blocked by the mTORC1 inhibitor rapamycin. ConclusionOur results demonstrate that NP-cGMP signaling activates mTORC1 via PKG, which is a component in the mechanism of adipose browning.

Natriuretic Peptides in Anxiety and Panic Disorder.

Natriuretic peptides exert pleiotropic effects on the cardiovascular system, including natriuresis, diuresis, vasodilation, and lusitropy, by signaling through membrane-bound guanylyl cyclases. In addition to their use as diagnostic and prognostic markers for heart failure, accumulating behavioral evidence suggests that these hormones also modulate anxiety symptoms and panic attacks. This review summarizes our current knowledge of the role of natriuretic peptides in animal and human anxiety and highlights some novel aspects from recent clinical studies on this topic.

Role of Phosphodiesterase 5 and Cyclic GMP in Hypertension.

Cyclic GMP (cGMP) is a ubiquitous intracellular second messenger that mediates a wide spectrum of physiologic processes in multiple cell types within the cardiovascular and nervous systems. Synthesis of cGMP occurs either by NO-sensitive guanylyl cyclases in response to nitric oxide or by membrane-bound guanylyl cyclases in response to natriuretic peptides and has been shown to regulate blood pressure homeostasis by influencing vascular tone, sympathetic nervous system, and sodium and water handling in the kidney. Several cGMPs degrading phosphodiesterases (PDEs), including PDE1 and PDE5, play an important role in the regulation of cGMP signaling. Recent findings revealed that increased activity of cGMP-hydrolyzing PDEs contribute to the development of hypertension. In this review, we will summarize recent research findings regarding the cGMP/PDE signaling in the vasculature, the central nervous system, and the kidney which are associated with the development and maintenance of hypertension.

Enhancement of cAMP-mediated inotropic responses by CNP is regulated differently by PDE2 in normal and failing hearts

Background Natriuretic peptide levels are increased in heart failure (HF). Atrial (ANP) and brain (BNP) natriuretic peptide mediate their effects preferentially through the natriuretic peptide receptor (NPR)-A, and C-type natriuretic peptide (CNP) through NPR-B. NPRs are membrane bound guanylyl cyclases that increase cyclic GMP (cGMP) production when activated. We have previously shown that NPR-B stimulation by CNP enhances b1-adrenoceptor (b1-AR)and 5-HT4 serotonin receptor-mediated signaling in failing hearts, probably through inhibition of phosphodiesterase (PDE) 3, a potential detrimental effect in the failing heart. In this study we examine the role of PDE2 in regulating the CNP-induced enhancement of b1-AR and 5-HT4 signaling in non-failing (Sham) and failing (HF) hearts, as PDE2 is a dual-selective PDE and can potentially be stimulated by cGMP.

Snake venom natriuretic peptides: Potential molecular probes

Natriuretic peptides (NPs) play a vital role in the regulation of pressure-volume homeostasis. They mediate vasodilation and increase water-electrolyte excretion through kidneys by activating membrane bound guanylyl cyclase receptors (NPR). Apart from the three mammalian isoforms, snake venom NPs with distinct pharmacological functions have been characterized. This study describes the molecular mechanism and physiological action of a novel NP from krait (Bungarus flaviceps) venom called KNP. This peptide has the conserved 17-residue NP ring with a 38-residue long C-terminal tail which has propensity to form an α-helix. Intravenous infusion of KNP in rats resulted in sustained and prolonged reduction in blood pressure without renal effects compared to the mammalian counterpart. It mediates vasodilatory function via endothelium in a NPR-independent manner. Deletion mutant studies show the presence of two active segments in KNP, namely; K-Ring and K-Helix. K-Ring functions similar to a classical NP; through its interaction with NPR, thereby evoking an endothelium-independent vasorelaxation response. Interestingly, K-Helix induces vasorelaxation through endothelium via NO-dependent pathways. Thus, despite the presence of a functional NP ring, the putative helical segment of KNP seems to drive the function of this NP. Further, infusion of K-Ring in rats shows similar vascular effects as mammalian NPs without diuresis. Despite the ability to activate NPR, K-Ring showed tissue-specific activity. Substitution of two amino acid residues in K-Ring restored renal function, suggesting the importance of conformational plasticity of NPs in activating downstream response. Thus, KNP provides a new paradigm in the understanding of NP biology and may open avenues for design and development of novel NP-based therapeutics.

Natriuretic Peptides and Cardiometabolic Health.

Natriuretic peptides are cardiac-derived hormones with a range of protective functions, including natriuresis, diuresis, vasodilation, lusitropy, lipolysis, weight loss, and improved insulin sensitivity. Their actions are mediated through membrane-bound guanylyl cyclases that lead to production of the intracellular second-messenger cyclic guanosine monophosphate. A growing body of evidence demonstrates that genetic and acquired deficiencies of the natriuretic peptide system can promote hypertension, cardiac hypertrophy, obesity, diabetes mellitus, the metabolic syndrome, and heart failure. Clinically, natriuretic peptides are robust diagnostic and prognostic markers, and augmenting natriuretic peptides is a target for therapeutic strategies in cardiometabolic disease. This review will summarize current understanding and highlight novel aspects of natriuretic peptide biology.

SGC-cGMP signaling: target for anticancer therapy.

The biologic endogenous production of cGMP was reported in the 1960s and followed by the demonstration of guanylyl cyclase activity and the isoforms of soluble and membrane-bound guanylyl cyclases. During the same period, cGMP specific phosphodiesterases also was discovered. Murad's lab established link between the endothelium derived relaxation factor (EDRF) and elevated cGMP concentration in the vascular system. October 12, 1998, the Nobel Assembly awarded the Nobel Prize in Medicine or Physiology to scientists Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries concerning nitric oxide (NO) as a signaling molecule in the cardiovascular system. In contrast with the short research history of the enzymatic synthesis of NO, the introduction of nitrate-containing compounds for medicinal purposes marked its 150th anniversary in 1997. Glyceryl trinitrate (nitroglycerin; GTN) is the first compound of this category. Alfred Nobel (the founder of the Nobel Prize) himself had suffered from angina pectoris and was prescribed nitroglycerin for his chest pain while he refused to take due to the induction of headaches. Almost a century after its first chemical use, research in the nitric oxide and 3',5'-cyclic guanosine monophosphate (NO/cGMP) pathway has dramatically expanded and the role of NO/cGMP in physiology and pathology has been extensively studied. Soluble guanylyl cyclase (sGC) is the receptor for NO. The α1β1 heterodimer is the predominant isoform of sGC that is obligatory for catalytic activity. NO binds to the ferrous (Fe(2+)) heme at histidine 105 of the β1 subunit and leads to an increase in sGC activity and cGMP production of at least 200-fold. In this chapter, we reviewed the studies of sGC-cGMP signaling in cell proliferation; introduced our work of targeting sGC-cGMP signaling for cancer therapy; and explored the role of sGC-cGMP signaling in the chromatin-microenvironment.

Krait natriuretic peptide (KNP): a non-classical NP

Background Cardiac homeostasis is a complex phenomenon, which is maintained by the interplay of factors that up and down regulate blood pressure. Natriuretic peptides (NPs), which cause vasodilation and increased waterelectrolyte excretion, are among the most vital hormonal controls of blood pressure. NPs elicit their function by binding to membrane bound guanylyl cyclase receptor. Three mammalian NPs are known, namely ANP, BNP and CNP, which are structurally similar with a 17 residue ring and a short (5-6 residues) C-terminal tail.

The effects of arginine and nitric oxide synthase inhibitors on intestinal damage in guanylyl cyclase‐G gene knockout mice with intestinal ischemia and reperfusion

Previous study demonstrated that membrane‐bound guanylyl cyclase (mGC)‐G gene knockout (KO) mice with intestinal ischemia and reperfusion (II/R) had significantly alleviated jejunal damage and apoptosis compared to wild type (WT) mice. It is known that apoptosis is controlled by guanylyl cyclase‐generated cGMP and arginine‐derived nitric oxide (NO). Therefore, we investigated the roles of NO and mGC‐G gene in II/R‐induced intestinal damage. mGC‐G KO mice suffered with 45 min intestinal ischemia were intra‐peritoneally administered with arginine (300 mg/kg) or inhibitors of nitric oxide synthase (NOS), i.e., aminoguanidine (100 mg/kg) or L‐NAME (10 mg/kg), and were executed after 3 or 24 hours of reperfusion. KO mice had significantly decreased jejunal mass and villus height and increased plasma IL‐6 and mucosal nitrate/nitrite (NOx) and cGMP compared to WT mice. However, II/R‐induced jejuanl atrophy and systemic and local inflammatory responses were attenuated in KO mice as shown in the less increases in plasma NOx and mucosal TNF‐alpha (p<0.05). At 3 hr reperfusion, arginine significantly increased plasma IL‐6 and mucosa TNF‐alpha; AMG increased mucosal TNF‐alpha; and L‐NAME increased plasma TNF‐alpha and mucosal cGMP in KO mice. At 24 hr reperfusion, arginine, AMG, and L‐NAME decreased mucosal TNF‐alpha, plasma IL‐6, and muscularis width, respectively. In addition, AMG and L‐NAME further decreased plasma NOx at 3 and 24 hr reperfusion. However, mGC‐G KO‐and II/R‐induced jejunal atrophy was not significantly altered by arginine or NOS inhibitors. These results suggest that mGC‐G may play certain roles in affecting the effects of NO on jejunal histology and inflammatory response (NSC 96–2320‐B‐309–003‐MY2).

C-Type Natriuretic Peptide Specifically Acts on the Pylorus and Large Intestine in Mouse Gastrointestinal Tract

C-type natriuretic peptide (CNP) exerts its main biological effects by binding to natriuretic peptide receptor B (NPR-B), a membrane-bound guanylyl cyclase receptor that produces cyclic guanosine monophosphate (cGMP). CNP is known to cause gastrointestinal (GI) smooth muscle relaxation. Experimental evidence suggests a connection between CNP signaling and GI function, with reactive regions in the GI tract possibly affecting transit; however, this relation has not yet been conclusively shown. Here, we show that CNP plays important region-specific roles in the GI tract of mice. We found that treatment with CNP (1 or 2 mg/kg) increased transient cGMP production in the pylorus, colon, and rectum, with the higher dose (2 mg/kg) enhancing gastric emptying in mice; this increase in cGMP levels was however absent in NPR-B-deficient short-limbed dwarfism (SLW) mouse. Furthermore, we found that NPR-B is highly expressed in the pylorus, colon, and rectum, being localized to nerve fibers and to the nuclei and cytoplasm of smooth muscle cells of the GI tract and blood vessels. Our invivo findings showed that NPR-B-mediated cGMP production after CNP administration specifically acted on the pylorus, colon, and rectum and contributed to gastric emptying. CNP may thus be a potential therapeutic agent for GI motility/transit disorders such as ileus and pyloric stenosis.

Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes

Crustacean hyperglycemic hormone (CHH) was originally identified in a neuroendocrine system-the X-organ/sinus gland complex. In this study, a cDNA (Prc-CHH) encoding CHH precursor was cloned from the hemocyte of the crayfish Procambarus clarkii. Analysis of tissues by a CHH-specific enzyme-linked immunosorbent assay (ELISA) confirmed the presence of CHH in hemocytes, the levels of which were much lower than those in the sinus gland, but 2 to 10 times higher than those in the thoracic and cerebral ganglia. Total hemocytes were separated by density gradient centrifugation into layers of hyaline cell (HC), semi-granular cell (SGC), and granular cell (GC). Analysis of extracts of each layer using ELISA revealed that CHH is present in GCs (202.8±86.7fmol/mg protein) and SGCs (497.8±49.4fmol/mg protein), but not in HCs. Finally, CHH stimulated the membrane-bound guanylyl cyclase (GC) activity of hemocytes in a dose-dependent manner. These data for the first time confirm that a crustacean neuropeptide-encoding gene is expressed in cells essential for immunity and its expression in hemocytes is cell type-specific. Effect of CHH on the membrane-bound GC activity of hemocyte suggests that hemocyte is a target site of CHH. Possible functions of the hemocyte-derived CHH are discussed.

Cyclic GMP generated by natriuretic peptide receptor B enhances β1-adrenoceptor signaling in normal and failing hearts

Background Atrial (ANP), B-type (BNP) and C-type (CNP) natriuretic peptide levels are increased in heart failure. Natriuretic peptides mediate their effects through natriuretic peptide receptors (NPRs), ANP and BNP preferentially through NPR-A and CNP through NPR-B. NPRs are membrane bound guanylyl cyclases that increase cyclic GMP (cGMP) production when activated. Increased cGMP levels may have beneficial cardiovascular effects through protein kinase G. In contrast, we have previously shown that NPR-B stimulation by CNP enhances b1-adrenoceptor (b1-AR) mediated signaling in failing hearts through inhibition of phosphodiesterase 3 (PDE3) [1]. This cardioexcitatory influence is longstanding and is thus probably detrimental in the failing heart. However, a comparison of the PDE3 inhibitory effect of NPR-B signaling in non-failing and failing hearts was not elucidated.

Behavioral Choice between Conflicting Alternatives Is Regulated by a Receptor Guanylyl Cyclase, GCY-28, and a Receptor Tyrosine Kinase, SCD-2, in AIA Interneurons ofCaenorhabditis elegans

Animals facing conflicting sensory cues make a behavioral choice between competing alternatives through integration of the sensory cues. Here, we performed a genetic screen to identify genes important for the sensory integration of two conflicting cues, the attractive odorant diacetyl and the aversive stimulus Cu(2+), and found that the membrane-bound guanylyl cyclase GCY-28 and the receptor tyrosine kinase SCD-2 regulate the behavioral choice between these alternatives in Caenorhabditis elegans. The gcy-28 mutants and scd-2 mutants show an abnormal bias in the behavioral choice between the cues, although their responses to each individual cue are similar to those in wild-type animals. Mutants in a gene encoding a cyclic nucleotide gated ion channel, cng-1, also exhibit the defect in sensory integration. Molecular genetic analyses suggested that GCY-28 and SCD-2 regulate sensory integration in AIA interneurons, where the conflicting sensory cues may converge. Genetic ablation or hyperpolarization of AIA interneurons showed nearly the same phenotype as gcy-28 or scd-2 mutants in the sensory integration, although this did not affect the sensory response to each individual cue. In gcy-28 or scd-2 mutants, activation of AIA interneurons is sufficient to restore normal sensory integration. These results suggest that the activity of AIA interneurons regulates the behavioral choice between the alternatives. We propose that GCY-28 and SCD-2 regulate sensory integration by modulating the activity of AIA interneurons.

Natriuretic peptide-sensitive guanylyl cyclase expression is down-regulated in human melanoma cells at simulated weightlessness

The membrane-bound guanylyl cyclases A and B (GC-A/B), which are receptors for natriuretic peptides, are expressed in cancer cells including melanomas and may represent new anticancer targets. Here, we report down-regulation of GC-A/B expression in human metastatic melanoma cells at simulated weightlessness in comparison to 1g conditions, suggesting attenuation of metastatic potential in weightlessness.

Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C

The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.

Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility

Echinoderm sperm use cyclic nucleotides (CNs) as essential second messengers to locate and swim towards the egg. Sea urchin sperm constitute a rich source of membrane-bound guanylyl cyclase (mGC), which was first cloned from sea urchin testis by the group of David Garbers. His group also identified speract, the first sperm-activating peptide (SAP) to be isolated from the egg investment (or egg jelly). This decapeptide stimulates sperm mGC causing a fast transient increase in cGMP that triggers an orchestrated set of physiological responses including: changes in: membrane potential, intracellular pH (pHi), intracellular Ca2+ concentration ([Ca2+]i) and cAMP levels. Evidence from several groups indicated that cGMP activation of a K+ selective channel was the first ion permeability change in the signaling cascade induced by SAPs, and recently the candidate gene was finally identified. Each of the 4 repeated, 6 trans-membrane segments of this channel contains a cyclic nucleotide binding domain. Together they comprise a single polypeptide chain like voltage-gated Na+ or Ca2+ channels. This new type of channel, named tetraKCNG, appears to belong to the exclusive club of novel protein families expressed only in sperm and its progenitors. SAPs also induce fluctuations in flagellar [Ca2+]i that correlate with changes in flagellar form and regulate sperm trajectory. The motility changes depend on [Ca2+]i influx through specific Ca2+ channels and not on the overall [Ca2+]i in the sperm flagellum. All cilia and flagella have a conserved axonemal structure and thus understanding how Ca2+ regulates cilia and flagella beating is a fundamental question.