All four subunits of HCN2 channels contribute to the activation gating in an additive but intricate manner.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that elicit electrical rhythmicity in specialized brain neurons and cardiomyocytes. The channels are dually activated by voltage and binding of cyclic adenosine monophosphate (cAMP) to their four cyclic nucleotide-binding domains (CNBDs). Here we analyze the effects of cAMP binding to different concatemers of HCN2 channel subunits, each having a defined number of functional CNBDs. We show that each liganded CNBD promotes channel activation in an additive manner and that, in the special case of two functional CNBDs, functionality does not depend on the arrangement of the subunits. Correspondingly, the reverse process of deactivation is slowed by progressive liganding, but only if four and three ligands as well as two ligands in trans position (opposite to each other) are bound. In contrast, two ligands bound in cis positions (adjacent to each other) and a single bound ligand do not affect channel deactivation. These results support an activation mechanism in which each single liganded CNBD causes a turning momentum on the tetrameric ring-like structure formed by all four CNBDs and that at least two liganded subunits in trans positions are required to maintain activation.