ABSTRACT The force-gated ion channel Piezo2 plays a crucial role in various mechanosensory functions, including light touch detection, proprioception, urination, blood pressure regulation, and lung inflation. Additionally, gain-of- function and loss-of function mutations have been implicated in Gordon syndrome, distal arthrogryposis, and overall deficits in proprioception. Given the crucial role of Piezo2 in physiologically significant mechanosensory processes and its direct association with human diseases, it is imperative to gain a mechanistic understanding of how Piezo2 functions as a sensor of mechanical stimuli. Although the physiological roles of Piezo2 are well understood, its fundamental properties—specifically, the tension-response relationship and gating kinetics— and how they both are affected by tissue-specific alternative splicing, are unknown. The overall objective of this proposal is to determine the fundamental biophysical properties of the human force-gated ion channel Piezo2 and systematically investigate six domains that undergo tissue-specific alternative splicing to determine their necessity and sufficiency in modifying function. My rationale is that knowing the biophysical properties of Piezo2 will enable us to understand its physiological roles in distinct tissues and cell types. I hypothesize that human Piezo2 and its splice variants exhibit distinct sensitivities and dynamic ranges in membrane tension sensing and differ in their gating kinetics. The scientific premise for this hypothesis is based on 1) the fact that Piezo2 is alternatively spliced in a tissue-specific manner, which implies an underlying difference in function, and 2) my preliminary data demonstrating distinct tension-response relationships in two Piezo2 splice variants. My research plan will determine the tension-response relationship (Aim1) and gating kinetics (Aim2) of human Piezo2 and the effects of its six spliced domains on these properties. The proposed research plan is innovative because it develops two innovative tools: Piezo2 constructs that allow for a structure-function investigation of spliced domains and a rigorous and unbiased image analysis program. It then applies these tools to explore two innovative concepts: quantification of the Piezo2 tension- response relationship and the hypothesis that spliced domains affect both this property and gating kinetics. The significance of this work is that it provides an understanding of the function and physiology of human Piezo2. This is essential for rationalizing the purpose of Piezo2 tissue-specific splicing, interpreting structural data of Piezo proteins, and comprehending the physiological forces, both in intensity and temporal dynamics, Piezo2 can sense or not. Its positive impact is that it will identify domains that selectively modulate Piezo2 function, which may become targets for treating Piezo-related diseases, such as itch, inflammatory pain, and chronic pain.