A mechanism of CALHM1 ion channel gating

The calcium homeostasis modulator (CALHM) proteins comprise a family of six genes, some of which have been demonstrated to function as ion channels. CALHM1, the founding member, is an extracellular Ca²⁺- and voltage-gated large-pore nonselective ion channel. The mechanisms by which Ca²⁺ and voltage regulate CALHM1 channel gating are unknown. Cryo-electron microscopic structures of CALHM1 and its paralogs have provided little insight into these features, although they have suggested that the amino-termini, including an amino-terminal helix (NTH) and the first transmembrane helix (TM1), may possess significant flexibility. Here, we investigated the role of the amino-terminus in the gating regulation of human CALHM1 channels expressed in <i>Xenopus</i> oocytes. Deletion of the NTH and the proximal end of TM1 markedly reduced the voltage dependence of channel gating, whereas extracellular Ca²⁺ retained the ability to close the channel, indicating that the amino-terminus is not the Ca²⁺-regulated gate. Furthermore, inhibition of channel currents by ruthenium red was independent of the presence of the amino-terminus and was mediated by effects on channel gating rather than pore block. The introduction of a cysteine residue into the proximal end of TM1 enabled complete inhibition of the channel by a cross-linking reagent under conditions in which the channel was in a closed state. Our findings indicate that although the NTH plays a role in voltage-dependent gating, it does not act as the gate itself. Instead, our results suggest that the gate in CALHM1 is formed by proximal regions of the first transmembrane domain.<b>NEW & NOTEWORTHY</b> CALHM1 is a voltage- and extracellular Ca²⁺-regulated large-pore ion channel that plays an essential role in taste perception. The mechanisms that regulate the opening and the closing of the channel are unknown. Here we explored the role of the amino-terminal region of the channel in gating regulation. Our data define the roles of the amino-terminus in channel gating, establishing components essential for the opening and closing of the CALHM1 channel gate.