Anne E. Carlson, Ph.D. - US grants

DESCRIPTION (provided by applicant): The human ether-a-go-go related gene 1 (Hergl) channel dysfunction plays a critical role in heart disease, a leading cause of death in the United States. Hergl is abundantly expressed in the heart, and its delayed rectifying potassium current contributes to the repolarization of the cardiac action potential. It is the bestcharacterized member of the ether-a-go-go (Eag) family of channels. We have analyzed the genetic sequence of Hergl, and other Eag family channels to identify two probable ligand-binding sites, and therefore characterize these channels as orphan receptors. The overall purpose of this proposal is to identify intracellular regulators for Hergl and other Eag family channels, and to characterize how these regulators modulate channel gating. Regulators will be identified with a high throughput chemical library screen of cellular metabolites, and inside-out patch clamp. Using molecular biology, protein biochemistry, and electrophysiology, I will determine how gating properties are affected by channel modulation and I will locate the site where the regulator binds the channel. The experiments in this proposal will contribute to our understanding of the role of these channels in physiology and pathology. Another benefit of these experiments is uncovering pathways involved in the central nervous system, such as learning and memory, or processes that underlie disease such as cardiac arrhythmia and cancer. Ultimately, this receptor site may be a potential drug target to ameliorate a number of diseases, including long QT syndrome. The goal of this study is to identify regulators for a family of proteins known as ether-a-go-go (Eag) channels, which are expressed throughout the brain and the heart. Mutations in the genes encoding these channels result in cardiac arrhythmias and death. Using a library screen of intracellular metabolites, I will identify intracellular regulators for these channels and determine how they affect these channels.

DESCRIPTION (provided by applicant): Fertilization is one of the most fundamental processes in nature, yet critical gaps exist in our understanding of this essential process. One of the earliest and most prevalent barriers to successful reproduction is the fertilization of an egg by more than one sperm, or polyspermy. This common problem, faced by the eggs of all sexually reproducing species, causes severe chromosomal defects and leads to embryonic mortality. This project will investigate the molecular mechanisms that ensure that each egg is fertilized by only one sperm, thus allowing for normal embryonic development. In the eggs of many organisms, fertilization evokes a prolonged membrane depolarization, which acts as a fast block to polyspermy. The fast polyspermy block requires the activity of one of more ion channel, but the molecular identity of any required channel is not known. In many species, including frogs, Cl- channels likely mediate this process. Coincidentally, a fertilization-induced increase i Ca2+ also occurs prior to the fast polyspermy block. A possible connection between these two events is the recently identified Ca2+ activated Cl- channel encoded by the TMEM16a gene. In specific aim one, I will identify the source of Ca2+ required for the depolarization at fertilizatin. Experiments outlined in specific aim two will uncover the role of the TMEM16a channel in the fast polyspermy block. Along with a fertilization-evoked increase in Ca2+, fertilization is also accompanied by a two-fold increase in phosphatidylinositol 4,5-bisphosphate (PIP2). The role that this elevated PIP2 may play in the first minutes after fertilization is unknown. Because PIP2 is a known regulator of structurally diverse ion channels and because the fertilization-evoked PIP2 elevation occurs within the time frame of the fast polyspermy block, I hypothesize that PIP2 regulates the fertilization-evoked depolarization. I will test this hypothesis in specific aim three and determine if PIP2 depletion affects the polyspermy block. The results of these experiments will contribute to our understanding of the biology of fertilization, and will provide the basis for future advances is reproductive medicine. PUBLIC HEALTH RELEVANCE: Fertilization is one of the most fundamental processes in nature, yet many of the processes involved in this process are unknown. Using cutting-edge experimental techniques, this project will uncover the molecular mechanisms that ensure that each egg is fertilized by only one sperm. These findings will contribute to our understanding of the events that begin new life.

The TMEM16a gene encodes for a Ca2+-activated Cl- channel that is broadly expressed in eukaryotes and plays an important role in human health and disease. TMEM16a channels are required for numerous physiologic processes, including regulation of neuronal and cardiac excitability, uterine contractility, regulation of electrolyte balance, and sensory transduction. Their importance is evidenced by the embryonic lethality of TMEM16a knockout mice, and by the association of mutations in this channel with craniofacial cancers. Aside from their Ca2+-dependent activation, it is not understood how these channels are regulated. Our lab is interested in uncovering the signaling mechanisms that modulate the activity of TMEM16a channels. Our goal is to understand the primary TMEM16a regulatory pathways, and to facilitate the development of novel therapeutic options for treating disorders caused by TMEM16a deficiencies.