Many human membrane proteins remain understudied, limiting opportunities for the development of novel therapeutics for currently incurable diseases. Our lab aims to uncover the physiological roles and molecular mechanisms of these proteins by combining expertise in structural biology, biochemistry, imaging, and genetics. Using C. elegans as a model organism, we systematically examine protein structure, interactome, expression patterns, and animal behavior associated with these targets. The advantages of C. elegans-including its simple yet highly conserved biology, ease of CRISPR-Cas9 genome editing, large-scale culture compatibility, transparent body for live imaging, and short life cycle—make it an ideal system for these investigations. Supported by NIGMS R01 funding, we have successfully determined the near-atomic resolution structures and elucidated the activation mechanisms of ATP-release channels (pannexins) and initiated studies on NIH-listed understudied membrane proteins linked to rare diseases, demonstrating the effectiveness of our approach. Over the next five years, we will focus on two other understudied membrane proteins with known implications in human diseases but poorly understood physiological roles and molecular mech- nisms. Our phased strategy begins with Phase I, where we will generate multiple C. elegans alleles using CRISPR-Cas9 genome editing and extrachromosomal arrays, alongside pre-cryo-EM studies to optimize protein purification conditions. In Phase II, we will conduct functional analyses and structural optimizations, employing label-free quantification mass spectrometry and RNA sequencing to identify signaling pathways and molecular functions associated with these proteins. Structural studies will focus on refining cryo-EM sample quality to ensure high-resolution data. In Phase III, we will leverage endogenously expressed proteins, which preserve their native conformations and complex formations, for advanced cryo-EM studies. Our overarching objective is to elucidate both the structures and molecular functions of these target proteins—a process that often works synergistically. Through our multidisciplinary approach, we aim to provide crucial insights into the physiological roles of these understudied membrane proteins, ultimately paving the way for future therapeutic advancements.