Single-channel recordings of coexpressed WT and IT channels. (A) Representative single-channel currents were recorded at −35 mV (left traces) or 0 mV (right traces), with openings represented by downward deflections from the closed state (c–) in symmetrical 250 mM KCl and 2 µM cis Ca²⁺ (left traces) and after the subsequent addition of 10 mM trans Ca²⁺ (right traces). Three traces of Group 3 channels recorded in the absence of 10 mM trans Ca²⁺ (left) indicate the absence of well-defined K⁺ conductance. Group 3 channels lack a Ca²⁺ current at 0 mV in the presence of 10 mM trans Ca²⁺. (B) Current–voltage relationships of Group 1 (•) and Group 2 (○) channels in 250 mM of symmetric KCl plus 10 mM trans Ca²⁺. (C) The boxed region indicated in B is expanded to highlight the negative shift in the reversal potential of Group 2 channels. (D) Proposed tetramer assembly model based on the assumption of independent WT and IT subunit assortment (left) and the expected reduction in fractional RYR1 Ca²⁺ conductance in muscle cells obtained from IT/+ and IT/IT mice (right), provided that Group 1 channels arise from tetramers with only one or fewer IT subunits, Group 2 channels from tetramers with two WT and two IT subunits, and Group 3 channels from tetramers with three or more IT subunits.