Human sperm KSper is physiologically activated by intracellular pH alkalization and CatSper-mediated Ca²⁺ signaling

The hyperpolarization of membrane potential (V_m) generated by the activation of the sperm-specific K⁺ channel (KSper) is considered as an important indicator for the evaluation of sperm-fertilizing capacity. However, owing to the relatively low pH sensitivity and low Ca²⁺ affinity of human KSper (hKSper), whether the changes of intracellular pH or cytosol Ca²⁺ ([Ca²⁺]_i) in response to physiological stimuli are sufficient to potentiate native hKSper is fairly obscure. Here, by utilizing quantitative V_m fluorometry and current-clamp recordings on human sperm, our results reliably demonstrated that physiologically relevant extracellular pH alkalization activated hKSper and evoked ∼20 mV hyperpolarization of V_m. Given that sperm Ca²⁺ influx is primarily mediated by the sperm-specific Ca²⁺ channel (CatSper), fluorometric results showed that progesterone, a physiological agonist of CatSper, remarkably hyperpolarized V_m in a dose-dependent manner. This hyperpolarizing effect (∼10-15 mV estimated by population V_m measurements) was largely suppressed by pharmacological inhibition of hKSper or the loss of functional CatSper. Surprisingly, electrophysiological recordings failed to detect progesterone-elicited hyperpolarization of V_m when employing Ca²⁺ chelator-free pipette solution. However, ∼10 mV hyperpolarization could be detected when the Ca²⁺ chelator contained in the pipette. In addition, [Ca²⁺]_i alteration under 100 nM could potently enhance hKSper activity, suggesting that hKSper can sense resting [Ca²⁺]_i levels. Taken together, our results clearly illustrate the activating effect of intracellular pH and [Ca²⁺]_i elevation on hKSper under physiological conditions, and moreover, broaden the understanding of the Ca²⁺-involved regulatory mechanism of hKSper.