Key points C‐type inactivation of voltage‐gated K+ channels is caused by a conformational change in the selectivity filter that prevents ion conductance. The role of subunit interaction during C‐type inactivation of hERG1 K+ channels was characterized by using concatenated tetrameric channels containing a defined composition and stoichiometry of wild‐type subunits and subunits with a mutation known to attenuate or accentuate inactivation gating. Analysis of the kinetics and voltage dependence of steady‐state inactivation for the concatenated channels indicated a variable extent of subunit interaction, dependent on the location of the mutation used to probe the gating process. Mutations located in the selectivity filter or pore helix disrupted inactivation in a dominant‐negative manner, suggesting that the final step of C‐type inactivation of hERG1 K+ channels is mediated by a concerted, highly cooperative interaction between all four subunits. AbstractAt depolarized membrane potentials, the conductance of some voltage‐gated K+ channels is reduced by C‐type inactivation. This gating process is voltage independent in Kv1 and involves a conformational change in the selectivity filter that is mediated by cooperative subunit interactions. C‐type inactivation in hERG1 K+ channels is voltage‐dependent, much faster in onset and greatly attenuates currents at positive potentials. Here we investigate the potential role of subunit interactions in C‐type inactivation of hERG1 channels. Point mutations in hERG1 known to eliminate (G628C/S631C), inhibit (S620T or S631A) or enhance (T618A or M645C) C‐type inactivation were introduced into subunits that were combined with wild‐type subunits to form concatenated tetrameric channels with defined subunit composition and stoichiometry. Channels were heterologously expressed in Xenopus oocytes and the two‐microelectrode voltage clamp was used to measure the kinetics and steady‐state properties of inactivation of whole cell currents. The effect of S631A or T618A mutations on inactivation was a graded function of the number of mutant subunits within a concatenated tetramer as predicted by a sequential model of cooperative subunit interactions, whereas M645C subunits increased the rate of inactivation of concatemers, as predicted for subunits that act independently of one another. For mutations located within the inactivation gate proper (S620T or G628C/S631C), the presence of a single subunit in a concatenated hERG1 tetramer disrupted gating to the same extent as that observed for mutant homotetramers. Together, our findings indicate that the final step of C‐type inactivation of hERG1 channels involves a concerted, all‐or‐none cooperative interaction between all four subunits, and that probing the mechanisms of channel gating with concatenated heterotypic channels should be interpreted with care, as conclusions regarding the nature of subunit interactions may depend on the specific mutation used to probe the gating process.