Calmodulin antagonists inhibit T-type Ca²⁺ currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction

The sperm acrosome reaction (AR) is a regulated exocytotic process required for gamete fusion. It depends on an increase in [Ca²⁺]_i mediated by Ca²⁺ channels. Although calmodulin (CaM) has been reported to regulate several events during the AR, it is not known whether it modulates sperm Ca²⁺ channels. In the present study we analyzed the effects of CaM antagonists W7 and trifluoroperazine on voltage-dependent T-type Ca²⁺ currents in mouse spermatogenic cells and on the zona pellucida-induced AR in sperm. We found that these CaM antagonists decreased T-currents in a concentration-dependent manner with IC₅₀ values of ∼10 and ∼12 μM, respectively. W7 altered the channels' voltage dependence of activation and slowed both activation and inactivation kinetics. It also induced inactivation at voltages at which T-channels are not activated, suggesting a promotion of inactivation from the closed state. Consistent with this, W7 inhibited the ZP-induced [Ca²⁺]_i transients in capacitated sperm. Likewise, W7 and TFP inhibited the AR with an IC₅₀ of ∼10 μM. In contrast, inhibitors of CaM-dependent kinase II and protein kinase A, as well as a CaM-activated phosphatase, had no effect either on T-currents in spermatogenic cells or on the sperm AR. Together these results suggest a functional interaction between CaM and the sperm T-type Ca²⁺ channel. They are also consistent with the involvement of T-channels in the AR.