Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling

Abstract

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...

Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect

“…3A). Recently, sAC protein was reported to be expressed in clear cells in the epididymis (26), however, because of the presence of endogenous ␤-galactosidase activity in the epididymis (22), we were unable to confirm this finding.…”

cAMP regulates plasma membrane vacuolar-type H + -ATPase assembly and activity in blowfly salivary glands

“…Evidence has been presented that cAMP activates an apical V-ATPase in Drosophila Malpighian tubules (39,40) and the gill epithelium of the crab Eriocheir sinensis (41). Moreover, pH regulation in rat epididymis is mediated by V-ATPase and regulated by a bicarbonate-activated soluble adenylyl cyclase (42). In all of these examples, however, the mode of V-ATPase regulation involves either the cycling of V-ATPase molecules between an intracellular vesicular pool and the plasma membrane (42) or is still undisclosed.…”

“…Moreover, pH regulation in rat epididymis is mediated by V-ATPase and regulated by a bicarbonate-activated soluble adenylyl cyclase (42). In all of these examples, however, the mode of V-ATPase regulation involves either the cycling of V-ATPase molecules between an intracellular vesicular pool and the plasma membrane (42) or is still undisclosed. Thus, we have identified the second messenger that controls reversible V-ATPase assembly in an animal cell.…”

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

“…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 .…”