Wijerathne Tharaka Darshana, Kim Jihyun, Yang Dongki, Lee Kyu Pil
Laboratory of Physiology, College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea.
Department of Physiology, College of Medicine, Gachon University, Incheon 21936, Korea.
Korean J Physiol Pharmacol. 2017 Mar;21(2):241-249. doi: 10.4196/kjpp.2017.21.2.241. Epub 2017 Feb 21.
Plasma membrane hyperpolarization associated with activation of Ca-activated K channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-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 BKCa activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 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 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. In contrast, elevation of intracellular Ca 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-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.
与钙激活钾通道激活相关的质膜超极化在受精过程中的精子获能中起重要作用。尽管已知Slo3（慢poke同源物3）与辅助γ2亚基LRRC52（富含亮氨酸重复序列的52）共同介导小鼠中对pH敏感的精子特异性钾电流KSper，但该通道在人类精子中的分子身份仍存在争议。在本研究中，我们测试了经典的大电导钙激活钾通道（BKCa）激活剂NS1619和LDD175对在HEK293细胞中与功能性相互作用的γ2亚基hLRRC52异源表达的人类Slo3的作用。如先前报道，Slo3钾电流不受埃博毒素或4-氨基吡啶的影响，但被20 mM四乙铵抑制约50%。细胞外碱化增强了hSlo3钾电流，而细胞内碱化和钙升高导致其激活电压向左偏移。在细胞内起作用以调节hSlo1门控的NS1619减弱了hSlo3钾电流，而LDD175增加了该电流并诱导膜电位超极化。在没有细胞内钙的情况下，LDD175诱导的增强与不同细胞内pH值（pH 7.3和pH 8.0）下半激活电压的变化无关。相反，细胞内钙的升高显著增强了LDD175诱导的hSlo3半激活电位的向左偏移。因此，其作用机制不涉及对hSlo3门控的pH依赖性调节；相反，LDD175可能调节hSlo3的钙依赖性激活。因此，LDD175可能激活天然的KSper，并可能在人类精子中诱导与膜超极化相关的超激活。
