PROJECT SUMMARY The goal of our proposed studies is to determine whether IDG-eligible CALHM4, CALHM5 and CALHM6 are ion channels or ion channel subunits. The CALHM (formerly FAM26) gene family is comprised of six homologs. We previously discovered the molecular mechanisms and physiological roles of CALHM1 and CALHM3 as novel ion channels. CALHM1 encodes a membrane protein expressed throughout the brain and in taste buds that lacks significant homology to other proteins, although five homologs have been identified with 20-44% sequence similarity, and CALHM proteins are conserved across species. We identified CALHM1 as a pore- forming subunit of an ion channel with a large pore-diameter and gating regulation by voltage and extracellular Ca2+ (Ca2+o). We discovered that CALHM1 is essential for perceptions of sweet, bitter and umami tastes by type II taste-bud cells, since CALHM1-knockout mice cannot perceive these tastants. We identified the essential role of CALHM1 by discovering that it is a voltage-gated ATP-permeable ion channel, and that tastant-evoked Na+ action potentials trigger ATP release as a neurotransmitter through CALHM1-associated channels to transduce taste information from the periphery to the central nervous system. We further discovered that whereas CALHM3 expression does not confer a novel ion channel, it is an essential component of the native voltage-gated ATP-release channel, contributing as a pore-forming subunit with CALHM1 to create a hetero-hexameric ATP-release channel in type II cells. Genetic deletion of CALHM3 also eliminates the ability of mice to perceive sweet, bitter and umami substances. These results suggest that other CALHM homologs are also ion channels or ion channel subunits, but there is no information regarding their molecular function. We hypothesize that CALHM4, CALHM5 and CALHM6 are ion channels, and furthermore that the CALHM family represents a family of ATP-release channels the contributes to purinergic signaling throughout the body. We will exploit our insights and technical approaches used to elucidate functions of CALHM1 and CALHM3 to determine whether CALHM4, CALHM5 and CALHM6 are ion channels or ion channel subunits. We will employ electrophysiology, optical imaging, ATP-release assays and biochemical approaches to test this overarching hypothesis. The impact of our research is expected to be the discovery of the functions of CALHM4, CALHM5 and CALHM6. Given the identification of CALHM1 and CALHM3 as ion- channel subunits, our findings are expected to provide information about whether CALHMs are a family of ion channel proteins. Identification of novel ion channels is expected to lead to new understanding of cell and tissue physiology, as our previous work in taste buds and taste perception did with the discoveries of the functions of CALHM1 and CALHM3. Accordingly, identification of the roles of the other CALHM homologs may inform new physiological insights and therapeutic opportunities.