2-APB-potentiated channels amplify CatSper-induced Ca(2+) signals in human sperm

Ca(2+)(i) signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca(2+)(i) signalling, stimulates a biphasic [Ca(2+)](i) rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca(2+)](i) transient was detectable in the anterior flagellum (where CatSper is expressed) 1–2 s before responses in the head and neck. Pre-treatment with 5 μM 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] ‘amplified’ progesterone-induced [Ca(2+)](i) transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca(2+)](i) response was unchanged. 2-APB (5 μM) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca(2+) accumulation downstream of the potentiated [Ca(2+)](i) transient. Pre-treatment with 50–100 μM 2-APB failed to potentiate the transient and suppressed sustained [Ca(2+)](i) elevation. When applied during the [Ca(2+)](i) plateau, 50–100 μM 2-APB caused a transient fall in [Ca(2+)](i), which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca(2+)](i) signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pH(i) (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca(2+) stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca(2+)](i) elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca(2+)](i) signals of human sperm.