The study investigates the temperature-dependent structural dynamics, ligand recognition, and gating mechanisms of TRPM4, a temperature-sensitive Ca²⁺-activated ion channel. By using single-particle cryo-electron microscopy at physiological temperatures (around 37 °C), researchers identified a 'warm' conformation distinct from the 'cold' conformation observed at lower temperatures. This warm conformation is driven by a temperature-dependent Ca²⁺-binding site in the intracellular domain and is essential for TRPM4 function under physiological conditions. The study also found that ligands such as decavanadate (DVT) and ATP bind to different locations of TRPM4 at physiological temperatures compared to lower temperatures, with these sites having functional relevance. The researchers captured structural snapshots of TRPM4 in different functional states, revealing a pre-open state or desensitized state at physiological temperatures. Additionally, they demonstrated that the temperature and Ca²⁺ synergistically trigger an upward swing of the intracellular domain, creating a new intersubunit interface and priming the DVT-binding site for interaction. The study highlights the importance of considering temperature as a critical factor in understanding the mechanisms of TRPM4 and other thermosensitive channels.The study investigates the temperature-dependent structural dynamics, ligand recognition, and gating mechanisms of TRPM4, a temperature-sensitive Ca²⁺-activated ion channel. By using single-particle cryo-electron microscopy at physiological temperatures (around 37 °C), researchers identified a 'warm' conformation distinct from the 'cold' conformation observed at lower temperatures. This warm conformation is driven by a temperature-dependent Ca²⁺-binding site in the intracellular domain and is essential for TRPM4 function under physiological conditions. The study also found that ligands such as decavanadate (DVT) and ATP bind to different locations of TRPM4 at physiological temperatures compared to lower temperatures, with these sites having functional relevance. The researchers captured structural snapshots of TRPM4 in different functional states, revealing a pre-open state or desensitized state at physiological temperatures. Additionally, they demonstrated that the temperature and Ca²⁺ synergistically trigger an upward swing of the intracellular domain, creating a new intersubunit interface and priming the DVT-binding site for interaction. The study highlights the importance of considering temperature as a critical factor in understanding the mechanisms of TRPM4 and other thermosensitive channels.