Inhibition of adenylyl cyclase by GTPase-deficient Gαi is mechanistically different from that mediated by receptor-activated Gαi.

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/ß5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/ß5 loop, the α4 helix, and the α4/ß6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment.

The role of soluble adenylyl cyclase in sensing and regulating intracellular pH.

Soluble adenylyl cyclase (sAC) differs from transmembrane adenylyl cyclases (tmAC) in many aspects. In particular, the activity of sAC is not regulated by G-proteins but by the prevailing bicarbonate concentrations inside cells. Therefore, sAC serves as an exquisite intracellular pH sensor, with the capacity to translate pH changes into the regulation of localization and/or activity of cellular proteins involved in pH homeostasis. In this review, we provide an overview of literature describing the regulation of sAC activity by bicarbonate, pinpointing the importance of compartmentalization of intracellular cAMP signaling cascades. In addition, examples of processes involving proton and bicarbonate transport in different cell types, in which sAC plays an important regulatory role, were described in detail.

The cAMP signaling module regulates sperm motility in the liverwort Marchantia polymorpha.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a universal signaling molecule that acts as a second messenger in various organisms. It is well established that cAMP plays essential roles across the tree of life, although the function of cAMP in land plants has long been debated. We previously identified the enzyme with both adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) activity as the cAMP-synthesis/hydrolysis enzyme COMBINED AC with PDE (CAPE) in the liverwort Marchantia polymorpha. CAPE is conserved in streptophytes that reproduce with motile sperm; however, the precise function of CAPE is not yet known. In this study, we demonstrate that the loss of function of CAPE in M. polymorpha led to male infertility due to impaired sperm flagellar motility. We also found that two genes encoding the regulatory subunits of cAMP-dependent protein kinase (PKA-R) were also involved in sperm motility. Based on these findings, it is evident that CAPE and PKA-Rs act as a cAMP signaling module that regulates sperm motility in M. polymorpha. Therefore, our results have shed light on the function of cAMP signaling and sperm motility regulators in land plants. This study suggests that cAMP signaling plays a common role in plant and animal sperm motility.

The Rectal Gland of the Shark: The Road to Understanding the Mechanism and Regulation of Transepithelial Chloride Transport.

Pictured, described, and speculated on, for close to 400 years, the function of the rectal gland of elasmobranchs remained unknown. In the late 1950s, Burger discovered that the rectal gland of Squalus acanthias secreted an almost pure solution of sodium chloride, isosmotic with blood, which could be stimulated by volume expansion of the fish. Twenty five years later, Stoff discovered that the secretion of the gland was mediated by adenyl cyclase. Studies since then have shown that vasoactive intestinal peptide (VIP) is the neurotransmitter responsible for activating adenyl cyclase; however, the amount of circulating VIP does not change in response to volume expansion. The humoral factor involved in activating the secretion of the gland is C-type natriuretic peptide, secreted from the heart in response to volume expansion. C-type natriuretic peptide circulates to the gland where it stimulates the release of VIP from nerves within the gland, but it also has a direct effect, independent of VIP. Sodium, potassium, and chloride are required for the gland to secrete, and the secretion of the gland is inhibited by ouabain or furosemide. The current model for the secretion of chloride was developed from this information. Basolateral NaKATPase maintains a low intracellular concentration of sodium, which establishes the large electrochemical gradient for sodium directed into the cell. Sodium moves from the blood into the cell (together with potassium and chloride) down this electrochemical gradient, through a coupled sodium, potassium, and two chloride cotransporter (NKCC1). On activation, chloride moves from the cell into the gland lumen, down its electrical gradient through apical cystic fibrosis transmembrane regulator. The fall in intracellular chloride leads to the phosphorylation and activation of NKCC1 that allows more chloride into the cell. Transepithelial sodium secretion into the lumen is driven by an electrical gradient through a paracellular pathway. The aim of this review was to examine the history of the origin of this model for the transport of chloride and suggest that it is applicable to many epithelia that transport chloride, both in resorptive and secretory directions.

The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

All-optical presynaptic plasticity induction by photoactivated adenylyl cyclase targeted to axon terminals.

Intracellular signaling plays essential roles in various cell types. In the central nervous system, signaling cascades are strictly regulated in a spatiotemporally specific manner to govern brain function; for example, presynaptic cyclic adenosine monophosphate (cAMP) can enhance the probability of neurotransmitter release. In the last decade, channelrhodopsin-2 has been engineered for subcellular targeting using localization tags, but optogenetic tools for intracellular signaling are not well developed. Therefore, we engineered a selective presynaptic fusion tag for photoactivated adenylyl cyclase (bPAC-Syn1a) and found its high localization at presynaptic terminals. Furthermore, an all-optical electrophysiological method revealed rapid and robust short-term potentiation by bPAC-Syn1a at brain stem-amygdala synapses in acute brain slices. Additionally, bPAC-Syn1a modulated mouse immobility behavior. These results indicate that bPAC-Syn1a can manipulate presynaptic cAMP signaling in vitro and in vivo. The all-optical manipulation technique developed in this study can help further elucidate the dynamic regulation of various cellular functions.

Behavior of Hypertrophied Right Ventricle during the Development of Left Ventricular Failure Due to Myocardial Infarction.

In order to determine the behavior of the right ventricle, we have reviewed the existing literature in the area of cardiac remodeling, signal transduction pathways, subcellular mechanisms, ß-adrenoreceptor-adenylyl cyclase system and myocardial catecholamine content during the development of left ventricular failure due to myocardial infarction. The right ventricle exhibited adaptive cardiac hypertrophy due to increases in different signal transduction pathways involving the activation of protein kinase C, phospholipase C and protein kinase A systems by elevated levels of vasoactive hormones such as catecholamines and angiotensin II in the circulation at early and moderate stages of heart failure. An increase in the sarcoplasmic reticulum Ca2+ transport without any changes in myofibrillar Ca2+-stimulated ATPase was observed in the right ventricle at early and moderate stages of heart failure. On the other hand, the right ventricle showed maladaptive cardiac hypertrophy at the severe stages of heart failure due to myocardial infarction. The upregulation and downregulation of ß-adrenoreceptor-mediated signal transduction pathways were observed in the right ventricle at moderate and late stages of heart failure, respectively. The catalytic activity of adenylate cyclase, as well as the regulation of this enzyme by Gs proteins, were seen to be augmented in the hypertrophied right ventricle at early, moderate and severe stages of heart failure. Furthermore, catecholamine stores and catecholamine uptake in the right ventricle were also affected as a consequence of changes in the sympathetic nervous system at different stages of heart failure. It is suggested that the hypertrophied right ventricle may serve as a compensatory mechanism to the left ventricle during the development of early and moderate stages of heart failure.

Selective enhancement of fear extinction by inhibiting neuronal adenylyl cyclase 1 (AC1) in aged mice.

Adenylyl cyclase 1 (AC1) is a selective subtype of ACs, which is selectively expressed in neurons. The activation of AC1 is activity-dependent, and AC1 plays an important role in cortical excitation that contributes to chronic pain and related emotional disorders. Previous studies have reported that human-used NB001 (hNB001, a selective AC1 inhibitor) produced analgesic effects in different animal models of chronic pain. However, the potential effects of hNB001 on learning and memory have been less investigated. In the present study, we found that hNB001 affected neither the induction nor the expression of trace fear, but selectively enhanced the relearning ability during the extinction in aged mice. By contrast, the same application of hNB001 did not affect recent, remote auditory fear memory, or remote fear extinction in either adult or aged mice. Furthermore, a single or consecutive 30-day oral administration of hNB001 did not affect acute nociceptive response, motor function, or anxiety-like behavior in either adult or aged mice. Our results are consistent with previous findings that inhibition of AC1 did not affect general sensory, emotional, and motor functions in adult mice, and provide strong evidence that inhibiting the activity of AC1 may be beneficial for certain forms of learning and memory in aged mice.

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid.

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3',5'-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 µm2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

A bioluminescent and homogeneous assay for monitoring GPCR-mediated cAMP modulation and PDE activity.

3',5'-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of Gαs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5'-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.

Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics.

Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gßγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.

ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering.

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Inhibition of adenylyl cyclase 8 prevents the upregulation of Orai1 channel, which improves cardiac function after myocardial infarction.

The upregulation of Orai1 and subsequent store-operated Ca2+ entry (SOCE) has been associated with adverse cardiac remodeling and heart failure (HF). However, the mechanism underlying Orai1 upregulation and its role in myocardial infarction remains unclear. Our study investigated the role of Orai1 in activating adenylyl cyclase 8 (AC8) and cyclic AMP (cAMP) response element-binding protein (CREB), as well as its contribution to cardiac dysfunction induced by ischemia and reperfusion (I/R). We found that I/R evoked an increase in the expression of Orai1 and AC8 in rats' hearts, resulting in a substantial rise in diastolic Ca2+ concentration ([Ca2+]i), and reduced ventricular contractions. The expression of Orai1 and AC8 was also increased in ventricular biopsies of post-ischemic HF patients. Mechanistically, we demonstrate that I/R activation of Orai1 stimulated AC8, which produced cAMP and phosphorylated CREB. Subsequently, p-CREB activated the ORAI1 promoter, resulting in Orai1 upregulation and SOCE exacerbation. Intramyocardial administration of AAV9 carrying AC8 short hairpin RNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i and improved cardiac contraction. Therefore, our data suggests that the axis composed by Orai1/AC8/CREB plays a critical role in I/R-induced cardiac dysfunction, representing a potential new therapeutic target to limit the progression of the disease toward HF.

Loss-of-function of GNAL dystonia gene impairs striatal dopamine receptors-mediated adenylyl cyclase/ cyclic AMP signaling pathway.

Loss-of-function mutations in the GNAL gene are responsible for DYT-GNAL dystonia. However, how GNAL mutations contribute to synaptic dysfunction is still unclear. The GNAL gene encodes the Gαolf protein, an isoform of stimulatory Gαs enriched in the striatum, with a key role in the regulation of cAMP signaling. Here, we used a combined biochemical and electrophysiological approach to study GPCR-mediated AC-cAMP cascade in the striatum of the heterozygous GNAL (GNAL+/-) rat model. We first analyzed adenosine type 2 (A2AR), and dopamine type 1 (D1R) receptors, which are directly coupled to Gαolf, and observed that the total levels of A2AR were increased, whereas D1R level was unaltered in GNAL+/- rats. In addition, the striatal isoform of adenylyl cyclase (AC5) was reduced, despite unaltered basal cAMP levels. Notably, the protein expression level of dopamine type 2 receptor (D2R), that inhibits the AC5-cAMP signaling pathway, was also reduced, similar to what observed in different DYT-TOR1A dystonia models. Accordingly, in the GNAL+/- rat striatum we found altered levels of the D2R regulatory proteins, RGS9-2, spinophilin, Gß5 and ß-arrestin2, suggesting a downregulation of D2R signaling cascade. Additionally, by analyzing the responses of striatal cholinergic interneurons to D2R activation, we found that the receptor-mediated inhibitory effect is significantly attenuated in GNAL+/- interneurons. Altogether, our findings demonstrate a profound alteration in the A2AR/D2R-AC-cAMP cascade in the striatum of the rat DYT-GNAL dystonia model, and provide a plausible explanation for our previous findings on the loss of dopamine D2R-dependent corticostriatal long-term depression.

Soluble adenylyl cyclase, the cell-autonomous member of the family.

Soluble adenylyl cyclase (sAC) is the evolutionarily most ancient of a set of 10 adenylyl cyclases (Adcys). While Adcy1 to Adcy9 are cAMP-producing enzymes that are activated by G-protein coupled receptors (GPCRs), Adcy10 (sAC) is an intracellular adenylyl cyclase. sAC plays a pivotal role in numerous cellular processes, ranging from basic physiological functions to complex signaling cascades. As a distinct member of the adenylyl cyclase family, sAC is not activated by GPCRs and stands apart due to its unique characteristics, regulation, and localization within cells. This minireview aims to honour Ulli Brandt, the outgoing Executive Editor of our journal, Biochimica Biophysica Acta (BBA), and longstanding Executive Editor of the BBA section Bioenergetics. We will therefore focus this review on bioenergetic aspects of sAC and, in addition, review some important recent general developments in the field of research on sAC.

Adenylate cyclase 3: a potential genetic link between obesity and major depressive disorder.

Obesity and major depressive disorder (MDD) are both significant health issues that have been increasing in prevalence and are associated with multiple comorbidities. Obesity and MDD have been shown to be bidirectionally associated, and they are both influenced by genetics and environmental factors. However, the molecular mechanisms that link these two diseases are not yet fully understood. It is possible that these diseases are connected through the actions of the cAMP/protein kinase A (PKA) pathway. Within this pathway, adenylate cyclase 3 (Adcy3) has emerged as a key player in both obesity and MDD. Numerous genetic variants in Adcy3 have been identified in humans in association with obesity. Rodent knockout studies have also validated the importance of this gene for energy homeostasis. Furthermore, Adcy3 has been identified as a top candidate gene and even a potential blood biomarker for MDD. Adcy3 and the cAMP/PKA pathway may therefore serve as an important genetic and functional link between these two diseases. In this mini-review, we discuss the role of both Adcy3 and the cAMP/PKA pathway, including specific genetic mutations, in both diseases. Understanding the role that Adcy3 mutations play in obesity and MDD could open the door for precision medicine approaches and treatments for both diseases that target this gene.

From membrane to nucleus: A three-wave hypothesis of cAMP signaling.

For many decades, our understanding of G protein-coupled receptor (GPCR) activity and cyclic AMP (cAMP) signaling was limited exclusively to the plasma membrane. However, a growing body of evidence has challenged this view by introducing the concept of endocytosis-dependent GPCR signaling. This emerging paradigm emphasizes not only the sustained production of cAMP but also its precise subcellular localization, thus transforming our understanding of the spatiotemporal organization of this process. Starting from this alternative point of view, our recent work sheds light on the role of an endocytosis-dependent calcium release from the endoplasmic reticulum in the control of nuclear cAMP levels. This is achieved through the activation of local soluble adenylyl cyclase, which in turn regulates the activation of local protein kinase A (PKA) and downstream transcriptional events. In this review, we explore the dynamic evolution of research on cyclic AMP signaling, including the findings that led us to formulate the novel three-wave hypothesis. We delve into how we abandoned the paradigm of cAMP generation limited to the plasma membrane and the changing perspectives on the rate-limiting step in nuclear PKA activation.

A molecular switch for neuroprotective astrocyte reactivity.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

Structural insights into membrane adenylyl cyclases, initiators of cAMP signaling.

Membrane adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. As effector proteins of G protein-coupled receptors and other signaling pathways, ACs receive and amplify signals from the cell surface, translating them into biochemical reactions in the intracellular space and integrating different signaling pathways. Despite their importance in signal transduction and physiology, our knowledge about the structure, function, regulation, and molecular interactions of ACs remains relatively scarce. In this review, we summarize recent advances in our understanding of these membrane enzymes.

The Role of Soluble Adenylyl Cyclase in the Regulation of Flagellar Motility in Ascidian Sperm.

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca2+-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca2+ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca2+ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion.