Structural and functional insights into large-pore channels

Project Summary The primary goal of this project is to enhance our understanding of the structures and functions of calcium homeostasis modulator (CALHM) proteins. These proteins play pivotal roles in diverse biological processes, ranging from neuromodulation and neuroinflammation to taste perception and neurodegeneration. CALHMs are large-pore channels facilitating the passage of ions and large molecules like ATP. Within the broader family of large-pore channels, CALHMs share classification with connexin, innexin, pannexin, and leucine-rich repeat- containing 8 (LRRC8). Dysfunctional CALHMs are implicated in neurological disorders, including Alzheimer's disease, depression, and neuroinflammation. Among the six CALHM members (1-6), CALHM1 has been unequivocally demonstrated to form voltage-sensitive channels permeable to ions and ATP. A polymorphism in calhm1 results in the Pro86Leu mutation, identified as a risk factor for Alzheimer's disease in various population groups. Genetic knockout studies in mice revealed that the absence of CALHM1 leads to a significant decline in learning and memory, underscoring its critical role in cerebral neuronal activities. Our research group has, thus far, accomplished the following milestones to promote the field of large-pore channels: (1) developed the insect cell expression technology, EarlyBac, for efficient expression of CALHM and other multi-heteromeric membrane proteins; (2) elucidated the structures of octameric chicken CALHM1 and undecameric human CALHM2 using single-particle electron cryomicroscopy (cryo-EM); (3) delineated the structure of human CALHM1, uncovering the interplay between the conserved lipid-binding pocket and channel activity; (4) disclosed the binding site and mode of an inhibitor, ruthenium red, at the CALHM1 channel pore; and (5) revealed the inaugural structure of the pannexin1 channel, elucidating the mechanism governing Cl- selectivity in the heptameric channel assembly, distinct from CALHM. However, the following specific gaps persist: (a) the absence of a clear structural basis for oligomeric states and function of CALHMs, (b) the dearth of structural and functional insights into ATP permeation in the CALHM1 channel; and (c) the lack of structural understanding regarding CALHM1/3 heteromers, known to exist physiologically in type-II taste cells and exhibit distinct voltage-sensitivity compared to CALHM1 homomers. Here we aim to address these shortcomings. Aim 1 will correlate oligomeric states of CALHM to channel functions by analyzing the structures and functions of the CALHM1-2 chimeric constructs. Aim 2 will uncover critical elements for ATP permeation in CALHM1. Aim 3 will reveal CALHM1/3 heteromer structures. These aims will be achieved through single-particle cryo-EM, and mechanistic hypotheses will be tested using patch-clamp electrophysiology and ATP flux assays. Successful completion promises significant strides in understanding CALHM structures and functions, paving the way for therapeutic strategies in neurological disorders.