Iberiotoxin and clofilium regulate hyperactivation, acrosome reaction, and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.