Na+/H+ Exchangers Involve in Regulating the pH-Sensitive Ion Channels in Mouse Sperm.

Hang Kang,Chen Chen,Na Zhao,Xu-Hui Zeng,Wei Zhang,Rong-Zu Huang,Min Liu

doi:10.3390/ijms22041612

Hang Kang, Chen Chen + Show 5 more

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https://doi.org/10.3390/ijms22041612

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Abstract

Sperm-specific K+ ion channel (KSper) and Ca2+ ion channel (CatSper), whose elimination causes male infertility in mice, determine the membrane potential and Ca2+ influx, respectively. KSper and CatSper can be activated by cytosolic alkalization, which occurs during sperm going through the alkaline environment of the female reproductive tract. However, which intracellular pH (pHi) regulator functionally couples to the activation of KSper/CatSper remains obscure. Although Na+/H+ exchangers (NHEs) have been implicated to mediate pHi in sperm, there is a lack of direct evidence confirming the functional coupling between NHEs and KSper/CatSper. Here, 5-(N,N-dimethyl)-amiloride (DMA), an NHEs inhibitor that firstly proved not to affect KSper/CatSper directly, was chosen to examine NHEs function on KSper/CatSper in mouse sperm. The results of patch clamping recordings showed that, when extracellular pH was at the physiological level of 7.4, DMA application caused KSper inhibition and the depolarization of membrane potential when pipette solutions were not pH-buffered. In contrast, these effects were minimized when pipette solutions were pH-buffered, indicating that they solely resulted from pHi acidification caused by NHEs inhibition. Similarly, DMA treatment reduced CatSper current and intracellular Ca2+, effects also dependent on the buffer capacity of pH in pipette solutions. The impairment of sperm motility was also observed after DMA incubation. These results manifested that NHEs activity is coupled to the activation of KSper/CatSper under physiological conditions.

Highlights

We demonstrated by patch-clamp recordings, Ca2+ fluorimetry, and motility measurements that the treatment of DMA remarkably attenuated the potentiation of KSper and CatSper channels through impairing the regulation of pHi, and thereby, reduced intracellular Ca2+ concentration and motility of sperm
DMA was reported as the potent inhibitor for Na+/H+ exchangers (NHEs) (IC50 = 6.9 μM) [25], whether the physiological activity of NHEs in mouse sperm was impaired by the application of DMA needs to be verified
By employing the pH indicator BCECF, it was found that 20 μM DMA acidified the cytoplasm of mouse sperm (Figure S1), which indicated the involvement of DMA-sensitive NHEs in the regulation of pHi

Summary

Introduction

Ion channels play critical roles in the regulation of cellular physiological activity. Ca2+ ion channel (CatSper), as two kinds of vital ion channels, were responsible for the modulation of membrane potential and Ca2+ influx, respectively [1,2,3,4,5,6,7,8]. The patients who exhibited the abnormal regulation of membrane potential primarily mediated by KSper or the mutation of CatSper-related gene might suffer from the infertile syndrome [16,17,18]. As the indispensability of KSper and CatSper channels in the fertilizing capacity of sperm, fully illuminating the physiological regulation of these two ion channels is, worth carrying out

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