Key points Intracellular Na+‐activated Slo2 potassium channels are in a closed state under normal physiological conditions, although their mechanisms of ion permeation gating are not well understood. A cryo‐electron microscopy structure of Slo2.2 suggests that the ion permeation pathway of these channels is closed by a single constriction of the inner pore formed by the criss‐crossing of the cytoplasmic ends of the S6 segments (the S6 bundle crossing) at a conserved Met residue. Functional characterization of mutant Slo2 channels suggests that hydrophobic interactions between Leu residues in the upper region of the S6 segments contribute to stabilizing the inner pore in a non‐conducting state. Mutation of the conserved Met residues in the S6 segments to the negatively‐charged Glu did not induce constitutive opening of Slo2.1 or Slo2.2, suggesting that ion permeation of Slo2 channels is not predominantly gated by the S6 bundle crossing. Abstract Large conductance K+‐selective Slo2 channels are in a closed state unless activated by elevated [Na+]i. Our previous studies suggested that the pore helix/selectivity filter serves as the activation gate in Slo2 channels. In the present study, we evaluated two other potential mechanisms for stabilization of Slo2 channels in a closed state: (1) dewetting and collapse of the inner pore (hydrophobic gating) and (2) constriction of the inner pore by tight criss‐crossing of the cytoplasmic ends of the S6 α‐helical segments. Slo2 channels contain two conserved Leu residues in each of the four S6 segments that line the inner pore region nearest the bottom of the selectivity filter. To evaluate the potential role of these residues in hydrophobic gating, Leu267 and Leu270 in human Slo2.1 were each replaced by 15 different residues. The relative conductance of mutant channels was highly dependent on hydrophilicity and volume of the amino acid substituted for Leu267 and was maximal with L267H. Consistent with their combined role in hydrophobic gating, replacement of both Leu residues with the isosteric but polar residue Asn (L267N/L270N) stabilized channels in a fully open state. In a recent cryo‐electron microscopy structure of chicken Slo2.2, the ion permeation pathway of the channel is closed by a constriction of the inner pore formed by criss‐crossing of the S6 segments at a conserved Met. Inconsistent with the S6 segment crossing forming the activation gate, replacement of the homologous Met residues in human Slo2.1 or Slo2.2 with the negatively‐charged Glu did not induce constitutive channel opening.