Celia M. Santi - US grants

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.

DESCRIPTION (provided by applicant): Sperm capacitation is a poorly understood series of molecular processes that occur in vivo in the female genital tract, and is absolutely essential to a sperm's ability to fertilize an egg. A key component of capacitation is the hyperpolarization of the sperm plasma membrane, but the molecular mechanism underlying hyperpolarization was unknown. We previously showed by using a SLO3 knock-out (SLO3 -/-) mouse that the SLO3 potassium channel is the main ion channel responsible for the hyperpolarization that occurs during sperm capacitation and that the SLO3 -/- is male infertile. We also demonstrated that sperm from SLO3 -/- mice have major deficits in sperm motility as well as in the Acrosome Reaction (AR). Since SLO3 channels are the major determinant of changes in membrane potential (Em) that occur during capacitation, it should now be possible to reveal how the components of the capacitating media trigger the activation of SLO3 channels and how SLO3 channel activation affects other signaling pathways that are active during capacitation and the AR. Since SLO3 -/- sperm have impaired motility and AR, and both events require increases in [Ca2+]i, we will investigate how SLO3 channel activation and subsequent changes in Em affect the [Ca2+]i changes that occur during capacitation and the AR. The overall objective of this proposal is to reveal the role of sperm membrane potential (Em) changes in capacitation and the AR. The SLO3 -/- mouse now represents an invaluable tool which permits us to gain greater experimental control over sperm Em and will reveal the importance of Em in each element of sperm physiology examined. The fact that SLO3 channels are essential to fertilization also suggests the possibility that genetic variation within this gene accounts for differences in fertility among male individuals. SLO3 channels also represent an ideal target for designing and testing blockers specific for this essential channel that can be used as contraceptive drugs. Finally, 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.

? DESCRIPTION (provided by applicant): Proper timing of delivery is important to the immediate and life-long health of both the newborn and the mother, but this event is often mistimed; in the U.S., approximately 12% of babies are born prematurely and up to 10% of pregnancies are described as post-term. For most of pregnancy, the uterus is maintained in a quiescent, non-contractile state in which the myometrial smooth muscle cells (MSMCs) are hyperpolarized, non-excitable, and quiescent. At term, the MSMCs become depolarized, excitable, and contractile. Currently, our limited understanding of how this transition is controlld hampers our ability to treat dysfunctional labor. Numerous ion channels are expressed in the MSMCs and contribute to regulation of uterine excitability. In particular, K+ channels play an important role in maintaining quiescence by controlling MSMC membrane potential by hyperpolarizing the membrane. Another key regulator in control of MSMC excitability is the hormone oxytocin, which binds to the oxytocin receptor (OTR), a G?q-coupled G-protein coupled receptor (G?qCR). As a result, Protein Kinase C (PKC) is activated and Ca2+ is released from intracellular stores, causing activation of actomyosin contraction. Additionally, it has been proposed that oxytocin triggers Ca2+ influx through voltage-dependent calcium channels by depolarizing the MSMC plasma membrane. However, the molecular mechanism responsible for this depolarization has not been established. Here, we propose to test the central hypothesis that the sodium-activated K+ channel SLO2.1 plays a key role in controlling the resting membrane potential of MSMCs and that its activity is down-regulated at term by either oxytocin-mediated inhibition or decreased expression, resulting in membrane depolarization. Several lines of evidence support this hypothesis. First, our preliminary data indicate that SLO2.1 is expressed in human MSMCs. Second, we report that SLO2.1 activity is modulated by oxytocin in both heterologous systems and MSMCs. Finally, SLO2.1 is known to be regulated by G?qCRs. The goals of this projects are the 1) define the temporal and spatial distribution of SLO2.1 channels in MSMCs, 2) investigate modulation of SLO2.1 channels by oxytocin; and 3) assess the contribution of SLO2.1 channels to regulation of uterine contractility. The research proposed here will establish the molecular pathways that regulate SLO2.1 activity, providing a biological basis for therapies designed to modulate uterine excitability.

Contraceptive methods; novel; Potassium Channel; Zidovudine;

PROJECT SUMMARY The high (~45%) rate of unintended pregnancies in the US is largely due to incorrect or inconsistent use of contraceptives, indicating that available contraceptives are failing to meet women's needs. An ideal female contraceptive will: 1) be highly effective at preventing pregnancy, 2) not act as an abortifacient, 3) have no negative side effects, and 4) not depend on hormones. We propose that the potassium (K+) channel SLO3 is an ideal target for the development of a contraceptive that meets these criteria. This idea is founded on several unique aspects of SLO3 channels. First, SLO3 is absolutely required for sperm capacitation; mice lacking SLO3 are healthy but infertile because their sperm fail to undergo processes essential to their ability to fuse with an oocyte, hyperactivation (a vigorous type of motility essential to fertilization) and the acrosome reaction (release of the acrosome content). Second, these processes occur in the female genital tract, so a drug targeting SLO3 will be an effective, non-hormonal, non-abortifacient, female contraceptive. Finally, SLO3 channels are only expressed in sperm cells in humans and other mammals, so a contraceptive targeting this channel will affect no other cell in a woman's body. Our objective here is to develop inhibitors of SLO3 that will act as non-hormonal and reversible female contraceptives. To achieve our objective, in the R61 Phase of the grant we will: 1) employ high-throughput screening (HTS) to identify potent and specific small-molecule inhibitors of SLO3 channels, and 2) perform patch clamp electrophysiology to test potency and selectivity of SLO3 inhibitors identified in aim 1. In the R33 Phase we will: 3) optimize SLO3 modulators via medicinal chemistry and 4) determine the effects of SLO3 inhibitors on human sperm K+ currents and human sperm function. The research proposed here will identify lead molecules that can be developed into an innovative class of female non-hormonal contraceptives that act by targeting sperm capacitation. The information obtained from these studies will also contribute new knowledge to the field, specifically a deeper understanding of the role of ion channels in sperm physiology.