Voltage-gated ion channels couple conformational change(s) of the voltage-sensing domain to those of the opening of an intracellular gate to allow ionic conduction. Much larger positive potentials are required to couple these conformational changes to the opening of the gate of Shaker K+ channels with the concurrent mutations V369I, I372L, and S376T (ILT) at the N-terminal end of the S4 segment. We used cut-open oocyte voltage clamp to study the biophysical and thermodynamical properties of heterotetrameric concatemerized channels with different stoichiometries of ILT mutations. The voltage-sensing domains of ILT mutant channels require smaller depolarization to activate but their intracellular gate does not immediately follow the movement of the voltage-sensing domain, requiring larger depolarization to open. Our results demonstrate that each subunit contributes equally to the rightward shift of the conductance–voltage relationship and that a single ILT-containing subunit is sufficient to induce a large enthalpic and entropic barrier, limiting opening of the intracellular gate.