Ligand-gated ion channels (LGICs) are regularly oligomers containing between two and five binding sites for ligands. Neither in homomeric nor heteromeric LGICs the activation process evoked by the ligand binding is fully understood. Here, we show on theoretical grounds that for LGICs with two to five binding sites, the cooperativity upon channel activation can be determined in considerable detail. The main requirements for our strategy are a defined number of binding sites in a channel, which can be achieved by concatenation, a systematic mutation of all binding sites and a global fit of all concentration–activation relationships (CARs) with corresponding intimately coupled Markovian state models. We take advantage of translating these state models to cubes with dimensions 2, 3, 4, and 5. We show that the maximum possible number of CARs for these LGICs specify all 7, 13, 23, and 41 independent model parameters, respectively, which directly provide all equilibrium constants within the respective schemes. Moreover, a fit that uses stochastically varied scaled unitary start vectors enables the determination of all parameters, without any bias imposed by specific start vectors. A comparison of the outcome of the analyses for the models with 2 to 5 binding sites showed that the identifiability of the parameters is best for a case with 5 binding sites and 41 parameters. Our strategy can be used to analyze experimental data of other LGICs and may be applicable to voltage-gated ion channels and metabotropic receptors.

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