The temperature-dependent response is mediated by a structurally diverse class of eukaryotic ion channels, many of which also exhibit polymodal behavior. To probe the foundational mechanisms of temperature-sensing, we characterized the temperature-dependent activation of a calcium-activated potassium channel, MthK, derived from archaebacteria isolated from hot springs. Functional complementation studies show that these channels are 100,000-fold more efficient at rescuing a potassium uptake deficient strain, LB2003, in low potassium agar plates at 36 °C than at 18 °C. By patch clamping giant E. coli spheroplasts, we show that MthK expressed in E.coli membranes is regulated by temperature. Electrophysiological activity of the purified MthK is extremely sensitive (Q10 ~100) to temperature change at low calcium concentrations, whereas channels lacking the calcium-sensing RCK domain are practically insensitive. Single-channel analysis of its allosteric behavior, which includes measurements under limiting conditions, shows that heat acts as an allosteric modifier by altering the coupling strength between the cytoplasmic calcium-sensing domain and pore domain. Our studies reveal a new mechanistic paradigm for allosteric regulation by physical stimuli. It is intriguing for us to understand how this coupling interaction undergoes a temperature-dependent process to activate the channel. Collaborated with Professor Vera Moiseenkova-Bell and Dr. Yaxian Zhao in the university of Pennsylvania, we purified MthK in a calcium-free buffer and solved a 4.8 Å resolution apo structure using cryo-EM. Comparing with the calcium bound structure (PDB: 6OLY), we observed a dramatic conformation change in the linker region upon calcium activation. However, how this structural change is related to the temperature activation requests further investigation. Superposing the isolated apo RCK domain (PDB:2OGU) with our structure, we observed a conformation regulation of the RCK domain by the pore. This result suggests the existence of coupling energy between the pore domain and the apo RCK domain. Moreover, helped with Dr. Vinay Idikuda and Dr. Vadim Klenchin, we screened over a million MthK mutants with complementation assay. Among these mutants, we achieved hundreds of cold-sensitive mutations in MthK. Through this study, we want to get a deeper understanding of the mechanism of temperature sensing in MthK.