Voltage-­gated calcium channels (VGCC) regulate the function of cells ranging from sperm to insulin-­producing pancreatic cells to neurons. There is growing recognition that specific lipids, sterols and the ganglioside GM1, regulate VGCC activity in health and disease including during fertilization, regulation of blood sugar, and pain sensation. However, determining mechanisms by which lipids regulate VGCC remains a major challenge in the field. This proposal addresses two questions of broad importance to biology & medicine: At a molecular level, how can membrane lipids regulate 1) VGCC activity and 2) cell function? Sperm are an outstanding model for these issues, because their ability to fertilize an egg is well-­known to be regulated by lipid dynamics. However, there is tremendous controversy regarding sperm calcium (Ca2+) channels, with electrophysiological recordings (at room temperature) only identifying the activity of a single, non-­VGCC channel (CatSper). In contrast, data from several groups using approaches of cell biology, genetics and pharmacology, show the activity of different channels including VGCC. Of importance, we found that sterols and GM1 regulate sperm VGCC activity in ways that typical electrophysiology practices can’t detect. This proposal represents a consortium of two laboratories, one with expertise in sperm electrophysiology, who have pioneered new methods to be able to detect channels regulated by lipids. The second laboratory was first to identify membrane lipid regulation of sperm VGCC, and has expertise in membrane lipid organization and function. Together, we propose to use new technologies and approaches to address this controversy, which is central to our understanding of sperm function & fertilization. This knowledge will empower clinicians to better understand the causes of male infertility, half of which are due to sperm function defects and are not detected by traditional semen analysis. Based on exciting preliminary data that clearly show evidence of more than one type of Ca2+ channel (using sperm from mice null for CatSper), as well as publications from both laboratories, we propose a series of experiments to investigate how lipids regulate mouse and human sperm VGCC and possibly other Ca2+ channels (Aim 1). These studies will utilize state-­of-­the-­art microscopy, pharmacology, mouse genetic models and electrophysiology under conditions that allow sperm membrane lipids to behave as they do in the female reproductive tract. We next propose to determine the precise molecular mechanisms by which sterols and GM1 regulate VGCC (Aim 2). To do this, we’ll express different VGCC subunits in specific cell lines, as well as artificial membrane systems, in which we can control both channel expression and the lipid composition. In both Aims, we will utilize innovative mouse strains either expressing genetically encoded Ca2+ indicators or lacking specific channel subunits. Together, these studies will provide broad mechanistic insight into the regulation of VGCC by membrane lipids, a matter of critical importance in both normal physiology as well as important disease states.