Significance Membrane receptors control innumerous essential functions of living cells and are proven drug targets for pharmacological interventions. Many receptors are composed of a number of subunits and contain multiple binding sites for ligands, resulting in intricate mechanisms of subunit activation. To understand these mechanisms, the subunit action is usually modeled by allosteric schemes assuming a fixed subunit cooperativity. Here, we use subunit concatenation, binding-site mutations, and global mathematical analyses to study the interaction of the individual subunits in heterooligomeric olfactory cyclic nucleotide-gated ion channels, which are composed of three types of subunits. Our analyses provide a detailed thermodynamic profile for these interactions upon channel activation, and they can be applied to any other oligomeric ion channel and receptor.