Spermatozoa undergo a poorly understood activation process induced by bicarbonate and mediated by cyclic adenosine 3',5'-monophosphate (cAMP). It has been assumed that bicarbonate mediates its effects through changes in intracellular pH or membrane potential; however, we demonstrate here that bicarbonate directly stimulates mammalian soluble adenylyl cyclase (sAC) activity in vivo and in vitro in a pH-independent manner. sAC is most similar to adenylyl cyclases from cyanobacteria, and bicarbonate regulation of cyclase activity is conserved in these early forms of life. sAC is also expressed in other bicarbonate-responsive tissues, which suggests that bicarbonate regulation of cAMP signaling plays a fundamental role in many biological systems.

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H ؉ ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.We recently identified bicarbonate-activated soluble adenylyl cyclase (sAC) 1 as a chemosensor mediating bicarbonate-dependent elevation of cAMP (1), defining a potential transduction pathway for cells to sense variations in bicarbonate, as well as the closely related parameters, pCO 2 and pH (1-3). sAC is distinct from transmembrane adenylyl cyclases. It is insensitive to regulation by forskolin or heterotrimeric G proteins (2) but is directly activated by bicarbonate ions. It does not have predicted transmembrane domains and is present in both soluble and particulate fractions of cellular extracts (4 -6). Mammalian sAC is similar to bicarbonate-regulated adenylyl cyclases present in cyanobacteria (1, 2), suggesting there may be a unifying mechanism for the bicarbonate regulation of cAMP signaling in many biological systems.sAC is highly expressed in spermatozoa (7) where it is proposed to mediate the bicarbonate-dependent cAMP elevation that precedes capacitation, hyperactivated motility, and acrosome reaction needed for fertilization (1). While spermatozoa mature and are stored along the epididymal lumen, they are kept in a quiescent state by an acidic pH of 6.5-6.8 and a low bicarbonate concentration of 2-7 mM (8). We have previously shown (9, 10) that a sub-population of epithelial cells, the so-called clear cells, are important players in the acidification capacity of the epididymis. Clear cells express high levels of the V-ATPase in their apical pole, and are responsible for the bulk of proton secretion in the vas deferens. Proton secretion by clear cells occurs in a chloride-independent but bicarbonate-dependent manner (11). Similarly to kidney intercalated cells, epididymal clear cells regulate their rate of proton secretion via V-ATPase recycling between intracellular vesicles and the apical plasma membrane (12). In these cells, as well a...

In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.The ubiquitous second messenger cAMP regulates a large variety of essential physiological processes such as gene expression, chromosome segregation and cellular metabolism. In mammalian cells, cAMP is synthesized by a family of nine transmembrane adenylyl cyclases (tmACs) and one sAC 1 . Unlike tmACs, which localize to the cellular membrane and respond to extracellular stimuli via heterotrimeric G proteins 1 , sAC is found in various intracellular compartments such as the mitochondria and the nucleus 2 . Its localization near intracellular cAMP targets is the impetus for current models of second messenger signal transduction, in which cAMP functions as a locally acting signaling molecule 2-4 .sAC is insensitive to the tmAC regulators calmodulin and heterotrimeric G proteins as well as the nonphysiological activator forskolin; instead, sAC senses physiological levels of bicarbonate 5 . Aside from its role as a universal physiological buffer maintaining cellular and extracellular pH, bicarbonate functions as a signaling molecule 3 , regulating many biological processes in mammals such as fertility 6 , acid-base homeostasis, breathing rate, metabolism and fluid transport (reviewed in ref. 7). As the only known signaling enzyme sensitive to physiological fluctuations of bicarbonate 5 , sAC probably mediates each of these processes. Bicarbonate activation of sAC is essential for sperm motility 8 as well as for pH-dependent COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. acid secretion in the epididymis and possibly the kidney 9 . In addition to its bicarbonate sensitivity, sAC is synergistically activated by calcium 10 , and this potentiation seems to be important for sperm maturation 11 . NIH Public AccessPrevious work has revealed the overall structure of tmAC enzymes and suggested a twometal ion mechanism for catalysis 12,13 . Despite their different regulation, mammalian sAC and tmACs are grouped into the nucleotidyl cyclase class III based on sequ...

"Soluble" adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V max of the enzyme with little effect on the apparent K m for substrate, ATPMg 2؉ . Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparent K m for ATP-Mg 2؉ . Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels. Two types of adenylyl cyclase (AC)1 are ubiquitously expressed in mammalian cells, a well characterized gene family of transmembrane ACs (tmACs) and the recently discovered "soluble" AC (sAC). The tmACs are plasma membrane bound, and their activities are regulated by G proteins in response to extracellular stimuli such as neurotransmitters and hormones (reviewed in Ref. 1). In contrast, sAC is associated with various intracellular organelles, including mitochondria, centrioles, mitotic spindle, mid-bodies, and nuclei (2). sAC activity is modulated by bicarbonate (3) and, as shown in this report, by Ca 2ϩ ; regulation by these intracellular signaling molecules suggests that sAC mediates cAMP-dependent responses to intrinsic cellular changes (4, 5).The catalytic mechanism of tmACs has been determined from biochemical and crystallographic studies. tmACs convert ATP to cAMP using two-metal catalysis where one ion acts as a free metal and the other coordinates ATP in the active site (6, 7). Its activators, G␣ s subunit or forskolin, stimulate tmACs by allosteric modulation of the active site (8, 9). More than 25 years ago, when soluble AC activity was first discovered, it was predicted to be molecularly distinct from tmACs because its activity appeared to be dependent on the presence of the divalent cation, Mn 2ϩ , and it was insensitive to forskolin and G protein regulation (10 -12). These differential properties enabled purification (13) and cloning of sAC from rat testis (14). The sAC gene is indeed molecularly distinct from tmACs; it possesses no transmembrane domains, and its catalytic domains are more closely related to those of cyanobacterial ACs than to those from other eukaryotic ACs. The purified soluble AC exhibited ϳ10-fold lower affinity for substrate ATP relative to tmACs (tmAC K m for ATP-Mn 2ϩ is ϳ 100...

Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.

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