Type II taste cells, which respond to sweet, bitter and umami tastants lack morphologically defined synapses. Instead, these taste cells utilize depolarization-evoked non-vesicular ATP release to communicate with afferent gustatory nerves. The overarching goal of the proposed studies is to determine how CALHM1 and CALHM3 contribute to a hetero-oligomeric ATP release channel in Type II taste cells. CALHM1, the pore forming subunit of an ion channel regulated by both voltage and extracellular Ca2+, is expressed in Type II taste cells and the brain. Calhm1 knockout mice cannot perceive sweet, bitter and umami tastants and exhibit defects in tastant-evoked ATP release from Type II taste cells. This suggests that CALHM1 plays an important physiological role in taste perception. However, there are differences in the kinetic properties of native CALHM1-dependent currents in Type II taste cells compared to CALHM1 currents in heterologous systems. It is important to identify the reasons for these inconsistencies to further our understanding of ATP release from Type II taste cells and taste perception. There are five additional Calhm family members of unknown function in humans. CALHM2 and CALHM3 are also expressed in Type II taste cells, but on their own, cannot form functional ion channels. Strikingly, co- expression of CALHM3 with CALHM1 in heterologous systems gave rise to a novel conductance with fast activation kinetics similar to those of the CALHM1-dependent currents observed in Type II taste cells. This led to the hypothesis that CALHM1 and CALHM3 may form a hetero-oligomeric ATP release channel in vivo. To determine the molecular basis for voltage-dependent gating of CALHM channels and define the role of CALHM3 in taste perception, a combination of electrophysiological and cell biological approaches will be utilized. CALHM proteins do not exhibit conserved architecture with canonical voltage-gated ion channels. To gain insight into the molecular determinants for the voltage-dependent gating of CALHM1, electrophysiology studies of chimeric channels and point mutants will be performed. This will lead to the identification of the activation gate and channel pore as well as specific residues required for voltage-dependent gating and ion selectivity properties. Knowledge gained from defining the molecular basis for CALHM1 gating will be critical for interpretation of similar electrophysiological studies that will be conducted o determine how CALHM3 contributes to the novel gating properties of the putative CALHM1 / CALHM3 hetero-oligomeric channel. To define the role of CALHM3 in taste perception as well as ATP release and CALHM1-dependent currents in Type II taste cells, behavioral, cell biological and electrophysiological analyses of Calhm3 knockout mice will be performed. This study will lead to an understanding of the molecular mechanism for CALHM channel gating and the role of CALHM3 in the perception of sweet, bitter and umami taste compounds.