External Ca 2+ acts upstream of adenylyl cyclase SACY in the bicarbonate signaled activation of sperm motility

Abstract

The HCO 3 − anion activates sperm motility, an important early step in capacitation, by increasing flagellar beat frequency through a pathway that requires the atypical adenylyl cyclase SACY and the sperm-specific Cα 2 catalytic subunit of PKA. Here we show that the accelerating action of HCO 3 − also requires the continued presence of external Ca 2+ (EC 50 ∼ 0.5 mM), and find that Ca 2+ can be replaced by Sr 2+ but not by Mn 2+. Ca 2+ is required for HCO 3 − to elevate cAMP, but not for cAMP-AM to increase beat frequency, indicating that external Ca 2+ acts before rather than after stimulation of SACY by HCO 3 −. With external Ca 2+ present, HCO 3 − does not alter cytosolic or near-membrane [Ca 2+]. Removal of external Ca 2+ initiates a slow decline in intracellular [Ca 2+] and rapid block of the HCO 3 −-evoked acceleration that is not relieved upon increasing internal [Ca 2+] by rapid photolysis of caged Ca 2+. We also find that the rapid ( t 1/2 ∼ 10 s) accelerating action of HCO 3 − is slowed more than three-fold by the carbonic anhydrase inhibitor acetazolamide. It is unaltered by the broad spectrum anion transport inhibitor SITS, and is not accompanied by detectable changes in intracellular pH. We propose that external Ca 2+ binds an unidentified extracellular protein that is required for HCO 3 − to engage cAMP-mediated activation of motility.