Functional Dimeric Organization of the Tetrameric HCN2 Pacemaker Channel

Abstract

Activation of HCN pacemaker channels is exerted by hyperpolarizing voltages and further promoted by the binding of cyclic nucleotides. The channels are tetramers and the molecular processes underlying channel activation are still unknown. We studied in voltage-activated channels the binding/unbinding of a fluorescent cAMP and the induced additional activation/deactivation simultanously using confocal patch-clamp fluorometry. The obtained current and fluorescence data were globally fitted in terms of a Markovian state model containing four binding steps in both the open and closed channel plus five closed-open isomerizations. The fit provided us a set of rate constants for all transitions which allowed us in turn to estimate all equilibrium association constants, KA, and also all equilibrium constants of the closed-open isomerizations, E. From these constants the respective Gibbs free energies (ΔG) were calculated. According to our model, the energy profile for the ligand-induced activation was considered for three distant ligand concentrations. As a result, the twofold liganded state produced an energy minimum in both the closed and the open channel. Hence, depending on the ligand concentration, states with zero, two, and four ligands are more stable than states with one or three ligands bound. This result leads us to propose that the homotetrameric HCN2 channel is functionally organized as a dimer. With respect to the equilibrium association constants, KA, there is positive cooperativity within the two dimers and negative cooperativity between the two dimers. A consequence of this molecular organization is that ligand-induced activation is not a four-step mechanism, as suggested by an apparently fourfold symmetric tetrameric channel, but only a two-step mechanism.