Abstract
The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.
Highlights
The Ca2+ channel CatSper represents the principal pathway for Ca21 entry into the flagellum of sperm for many species [1,2,3,4,5,6,7]
To scrutinize whether CatSper is activated by intracellular cAMP or cAMP/protein kinase A (PKA) signaling, we studied the action of bicarbonate, IBMX, and adenosine in human sperm
The amplitudes were similar in the absence and presence of cAMP (1 mM) in the pipette solution (Fig. 1, D and E): CatSper inward currents at 280 mV were 220.7 6 8.4 pA (n = 50) and 223.6 6 11.9 pA (n = 16); mean outward currents at 180 mV were 53.3 6 16.2 pA (n = 50) and 58.7 6 21.4 pA (n = 16), respectively; subtracting the extracellular solution containing Mg21 and Ca21 (HS)-leak currents had no bearing on the results (Fig. S1A), and cAMP did not affect the kinetics of CatSper gating, either (Fig. S1B)
Summary
To scrutinize whether CatSper is activated by intracellular cAMP or cAMP/PKA signaling, we studied the action of bicarbonate, IBMX, and adenosine in human sperm. The intracellular cAMP level and activation of the cAMP/PKA-signaling pathways seem not to modulate stimulus-induced gating of human CatSper either: bicarbonate and IBMX did not affect CatSper evoked by stimulation with a buffer of pH 8.6 and containing a high [K1], respectively (Fig. 1, I–L). We identified the pH of bicarbonate-containing buffers as a potential source of artifacts: the pH of buffers containing bicarbonate is tied to the partial pressure of CO2 [57] and, unstable at ambient conditions To illustrate this issue, we monitored the pH of human tubal fluid (HTF) exposed to ambient air in the absence and presence of bicarbonate using a fluorescent pH indicator. In sperm loaded with a fluorescent pH indicator, air-exposed (.60 min) bicarbonate-HTF evoked an alkalization of the pHi that was similar to the pHi alkalization evoked by alkaline (pH 7.8) HTF lacking bicarbonate (HTF7.8) or by the weak base ammonium chloride (NH4Cl)
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