Isolation and characterization of the primary structure of testis-specific L-type calcium channel: implications for contraception.
Isolation and characterization of the primary structure of testis-specific L-type calcium channel: implications for contraception.
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DOI：
10.1093/molehr/3.3.255
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发表时间：
1997-03
影响因子：
4
通讯作者：
Leslie O. Goodwin;N. Leeds;I. Hurley;F. Mandel;Robert G. Pergolizzi;S. Benoff
中科院分区：
文献类型：
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作者：
Leslie O. Goodwin;N. Leeds;I. Hurley;F. Mandel;Robert G. Pergolizzi;S. Benoff
钙通道阻断药物的治疗性给药与甘露糖受体表达减少和医源性人类男性不育症相关。在本报告中，我们研究了二氢吡啶的药理活性是否会导致不孕状态的产生，二氢吡啶可阻止钙通过电压依赖性钙通道流入。细胞外钙的流入是人类精子启动孕酮刺激顶体反应的绝对必要条件。为了确定二氢吡啶是否可以抑制黄体酮诱导的顶体丢失，我们研究了大鼠和人类精子中表达的一种蛋白质，该蛋白质在抗原上和cDNA序列上都与大鼠心肌电压依赖性钙通道的α1亚基相关，该亚基形成了通道的孔。使用逆转录聚合酶链式反应，我们从大鼠睾丸 mRNA 中分离出一个 2169 个碱基的克隆，其序列与大鼠心肌钙通道的 α1 亚基的序列基本相同，但由于剪接和交替外显子使用而有 84 个碱基的变化。这种变化插入了编码两亲性跨膜螺旋的肽盒，该螺旋构成骨骼肌钙通道离子孔的一部分，调节钙通道激活的动力学，并且可以充当膜内二氢吡啶结合位点。与此同时，来自有生育能力的捐献者的人类精子在体外暴露于硝苯地平。硝苯地平在可兴奋细胞中有效的浓度下抑制黄体酮刺激的钙内流和随后的人类精子顶体反应，但需要较长时间才能实现。相反，孕酮配体结合不受硝苯地平治疗的影响。这些数据表明人类精子表达对硝苯地平有反应的 L 型钙通道。假设精子钙转运途径高度保守，则获得人类精子通道阻断的缓慢动力学可以与通道激活和电导的变化相关，而通道激活和电导与大鼠中观察到的交替剪接产生的亚型多样性相关。这些数据为大鼠和人类精子中存在功能性 L 型电压依赖性钙通道提供了明确的证据。这些数据还定义了该通道中钙进入拮抗剂的改变的结合位点，并为新型男性避孕药的设计提供了独特的目标。
Therapeutic administration of calcium channel-blocking medications has been correlated with reduced mannose receptor expression and iatrogenic human male infertility. In this report, we investigate whether the pharmacological activity of dihydropyridines, which block calcium influx through voltage-dependent calcium channels, contributes to the production of an infertile state. An influx of extracellular calcium is an absolute requirement for the initiation of a progesterone-stimulated acrosome reaction by human spermatozoa. To determine whether dihydropyridines could inhibit progesterone-induced acrosome loss, we have studied a protein expressed in rat and human spermatozoa which is related both antigenically and by cDNA sequence to the alpha 1 subunit of the rat cardiac muscle voltage-dependent calcium channel, which forms the pore of the channel. Using reverse transcription-polymerase chain reaction, we have isolated a 2169 base clone from rat testis mRNA whose sequence was largely identical to that of the alpha 1 subunit of the rat cardiac muscle calcium channel, but had an 84 base change, attributable to splicing and alternate exon usage. This change inserts a peptide cassette encoding an amphipathic membrane-spanning helix that constitutes part of the ionic pore of the skeletal muscle calcium channel regulating the kinetics of activation of the calcium channel and may serve as an intramembrane dihydropyridine binding site. In parallel, human spermatozoa from fertile donors were exposed to nifedipine in vitro. Nifedipine inhibited progesterone-stimulated calcium influx and subsequent acrosome reactions in human spermatozoa at concentrations effective in excitable cells, but required a prolonged time to do so. In contrast, progesterone ligand binding was unaffected by nifedipine treatment. These data demonstrate that human spermatozoa express an L-type calcium channel which is responsive to nifedipine. Assuming sperm calcium transport pathways are highly conserved, the slow kinetics by which the blockade of the human sperm channel was obtained can be correlated with alterations in channel activation and conductance associated with isoform diversity generated by alternate splicing as observed in the rat. These data provide unequivocal evidence for the presence of functional L-type voltage-dependent calcium channels in rat and human spermatozoa. The data also define an altered binding site for calcium entry antagonists in this channel and offer a unique target for the design of new male contraceptive agents.