Molecular Basis of a Calcium Sensor Underlying Synaptic Plasticity, Working Memory, and Behavior

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Project Abstract/Summary: In order for the brain to store and process information, the connections between neurons must be able to change in response to use and previous activity patterns. This process is known as synaptic plasticity. Understanding the fundamental mechanisms of synaptic plasticity is important as we seek to gain a mechanistic understanding of the molecular processes underlying behavior, memory, and information processing. One form of synaptic plasticity is paired-pulse facilitation (PPF), which is defined as an increased probability of neurotransmitter release in response to the second stimulus of two closely spaced stimuli. Although heavily studied, the mechanisms underlying PPF remain unclear. It has long been speculated that PPF is a result of calcium remaining bound to a calcium sensor prior to the second stimulus of two closely spaced stimuli. In other words, PPF is a function of how fast calcium comes off of the sensor. The identity of this sensor has eluded neuroscientists for decades. My preliminary data and several high profile studies show that the protein synaptotagmin 7 (syt7) is required for PPF making it the most likely candidate sensor. The calcium sensing ability of the synaptotagmin family of proteins arises from their two C2 domains and two calcium binding loops per domain. To date, however, there is no evidence that syt7 plays a direct role in PPF as simply knocking out syt7 is insufficient because syt7 may be the direct player or just enable some other factor's function. Based on the idea that PPF is a function of the calcium off rate of the sensor, I hypothesize that increasing/decreasing the calcium off rate of syt7 will respectively /narrow the space between stimuli or the time course of PPF. Therefore, the objective of this proposal is to provide the first direct evidence of whether syt7 is the proximal sensor for PPF. This will be achieved by generating and characterizing mutant forms of syt7 with altered calcium binding kinetic properties (Aim 1). Our lab has an established history of tuning the kinetics of other calcium sensors. We will also directly assess whether syt7 is the proximal sensor for PPF by expressing syt7 calcium off rate mutants in syt7 knockout neurons to narrow/widen the PPF tuning window (Aim 2). Successful completion of this proposal will provide direct evidence whether syt7 is the proximal sensor for PPF. If syt7 is the sensor, this study will provide new tools that could alter the time course of PPF to study how PPF functions in neuropsychiatric diseases, behavior, information processing, memory, and other disease states. If tuning the kinetics of syt7 does not result in concomitant changes in the PPF tuning window, our work will rule out syt7 as the proximal calcium sensor for PPF, and will motivate entirely new lines of study to understand the syt7 requirement for this form of plasticity. Additionally, the proposed studies provide me with valuable training specifically designed to guide me toward my overall career goal of becoming a physician-scientist principal investigator.