Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BK_Ca activator LDD175

Plasma membrane hyperpolarization associated with activation of $Ca^{2+}$-activated $K^+$ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary ${\gamma}^2$-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, sperm-specific $K^+$ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical $BK_{Ca}$ activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting ${\gamma}^2$ subunit, hLRRC52. As previously reported, Slo3 $K^+$ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 $K^+$ current, and internal alkalinization and $Ca^{2+}$ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 $K^+$ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular $Ca^{2+}$. In contrast, elevation of intracellular $Ca^{2+}$ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate $Ca^{2+}$-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.