A NOVEL PH DEPENDENT POTASSIUM CHANNEL IN MAMMALIAN SPERM

描述(申请人提供)：SLO_3通道是高电导SLO钾(K+)通道家族的成员，由电压和细胞内pH(Phi)激活。SLO_3通道仅在哺乳动物中发现，并且仅位于睾丸中。我们发现slo3是一种快速进化的基因，就像许多控制男性生殖的基因一样。我们的初步结果表明，SLO_3存在于成熟精子中，并受蛋白激酶A(PKA)的调节。我们将结合免疫组织化学、遗传学、分子生物学和电生理学来研究这种特殊的离子通道在精子生理中的作用。为了进一步阐明它在生育中的作用，我们将在小鼠中产生并分析SLO_3通道的基因敲除(K/O)。Slo3基因的敲除将使我们能够通过比较野生型和突变型动物中存在的K+电流来验证我们在成熟精子和精母细胞中观察到的K+电流的身份。SLO_3基因的敲除也将使我们能够确定该通道在精子生理和行为中的功能作用。因此，我们将从K/O小鼠的生育能力、精子细胞的产生、精子活力、精子获能、顶体反应和精子体积调节等方面来研究SLO_3通道缺失所导致的表型。这些研究的意义在于：1.本研究将有助于我们理解K+通道在精子运动、获能、顶体反应和关键渗透调节等重要生理事件中的作用。 2.对SLO_3通道的研究有助于体外受精(IVF)技术。了解精子的电学性质和外部离子环境的基本方面可能对提高临床试管受精程序的效率具有重要意义。3.SLO_3通道可能是男性避孕的药理靶点。4.Slo3基因的遗传变异可能影响男性的生育能力。与公共卫生相关：快速进化的slo3基因编码一种只在哺乳动物中发现的高电导K+通道，并且仅位于男性生殖细胞中。我们的初步结果表明，phi和电压都可以激活精子，并可能受到PKA的调节，这些特性表明在哺乳动物精子生理学中起着关键作用。利用各种分子、生理和基因敲除技术，我们将揭示这些离子通道的功能及其在受精过程中的作用，这些信息可能会影响体外受精和避孕领域。

DESCRIPTION (provided by applicant): The SLO3 channel is a member of the high conductance SLO potassium (K+) channel family and is activated by both voltage and intracellular pH (pHi). The SLO3 channel is found only in mammals and is located exclusively in testis. We found that slo3 is a rapidly evolving gene like many genes that govern male reproduction. Our preliminary results show that SLO3 is present in mature sperm and is modulated by protein kinase A (PKA). We will use a combination of immunohistochemistry, genetics, molecular biology and electrophysiology to study the role of this particular ion channel in the physiology of the sperm. To further elucidate its role in fertility we will generate and analyze a gene knock-out (K/O) of SLO3 channels in mouse. A knock-out of the Slo3 gene will allow us to verify the identity of the K+ current that we observe in mature sperm and spermatocytes by comparing the K+ currents present in wild-type and mutant animals. A SLO3 knock-out will also allow us to determine the functional role of the channel in sperm physiology and behavior. Thus, we will examine the phenotype resulting from the loss of SLO3 channels with respect to the fertility of the K/O mouse, the production of sperm cells, sperm motility, sperm capacitation, the acrosome reaction and volume regulation of sperm. The significance of these studies are: 1. This study will contribute to our understanding of the role that K+ channels play in critically important events in sperm physiology such as motility, capacitation, the acrosome reaction and crucial osmotic control. 2. Studies of SLO3 channels may contribute to in vitro fertilization (IVF) techniques. Knowledge of the electrical properties of sperm and the essential aspects of the external ionic environment may have important implications for improving the efficiency of clinical IVF procedures. 3. SLO3 channels may be a pharmacological target useful in male contraception. 4. Genetic variation in the Slo3 gene may affect male fertility. PUBLIC HEALTH RELEVANCE: The rapidly evolving slo3 gene encodes a high conductance K+ channel found only in mammals and located exclusively in male germ cells. Our preliminary results show activation by both pHi and voltage, and its probable modulation by PKA, properties which suggest a key role in mammalian sperm physiology. Using a variety of molecular, physiological, and gene knock-out techniques we will reveal the function of these ion channels and their involvement in fertilization, information that may impact the field of in vitro fertilization and contraception.