Journal of Biological Chemistry

The hyperpolarization of membrane potential (Vm) 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 Vm 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 Vm. 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 Vm in a dose-dependent manner. This hyperpolarizing effect (∼10−15 mV estimated by population Vm 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 Vm 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.

human sperm–specific K+ channel

hKSper

membrane potential

pH alkalization

Ca2+ signaling

sperm-specific Ca2+ channel

CatSper

resting [Ca2+]i level

1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid

intracellular Ca2+

sperm-specific Ca2+ channel

sperm-specific potassium channel

mouse slowpoke homologous 3

© 2025 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biologyé