How to Define Cooperativity for the Ligand-Induced Gating of HCN2 Channels?

Abstract

The term ‘cooperativity’ is generally used to functionally characterize enzymes and receptors with multiple binding sites when the affinity at one binding site influences the affinity at the other binding site(s). The affinity increase for the primary ligand is a simple energetic consequence of its action because the energy used for a confomational change must be provided by the binding itself. The affinity change in the other binding sites is a speciality of the multimeric protein, determining its characteristic properties. An affinity increase of the subsequent binging sites is usually termed ‘positive cooperativity’ and an affinity decrease ‘negative cooperativity’. Positive cooperativity has been often described by the Monod-Wyman-Changeux model, assuming a constant allosteric factor for the successive affinity increase for each binding step. Using confocal patch-clamp fluorometry and a fluorescence-labelled cAMP, we recently determined for HCN2 channels the rate constants in a Markovian model containing four sequential binding steps, in both the closed and the open channel, and five closed open isomerizations. The equilibrium association constants of the binding steps, i.e. the affinity of the binding sites, changed with the sequence 1.5×106, 9.0×106, 1.2×104, 2.6×106 (M−1), resulting in a sequence of cooperativity of ‘positive-negative-positive’. However, cooperativity can also be related to the rates of binding and unbinding, i.e. to physical processes proceeding in time. If considering, for example, the binding rates in the closed channel, the sequence is 5.4×106, 8.4×105, 9.9×104, 2.2×107 (M−1s−1), resulting in a sequence of cooperativity of ‘negative-negative-positive’. In the open channel this sequence is similar. As a conclusion we propose to distinguish between cooperativity related to the affinities of the binding sites from cooperativity related to the rates of binding or unbinding at the binding sites, providing more precise information about the underlying physical processes.