How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels

Abstract

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.

Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1

“…To the best of our knowledge, our study provides the first structural indication that CNBDs and voltage sensors interact directly, although this was proposed recently for HCN channels based on functional assays51. Our structures show that in the presence of cAMP, the VSDs are slightly lower in the membrane and their helices are tilted.…”

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

“…Negative binding cooperativity has previously been suggested for full-length CNG and HCN eukaryotic channels based on patch clamp fluorometry experiments (11)(12)(13)(14). Similarly, binding curves for cAMP to the isolated CNBDs of both HCN2 and HCN4 suggest negative cooperativity, with the first cAMP molecule binding with a 10-fold higher affinity than the subsequent molecules (48).…”

“…Current understanding largely derives from electrophysiological experiments on cells heterologously expressing these channels (1,10). Recent technical advances in confocal patch clamp fluorometry permit concomitant ligand-binding measurements and current recordings in the same membrane patch, allowing for detailed analysis of binding cooperativity and activation states triggered by partial ligand binding (11)(12)(13)(14). For example, Kusch et al (12) provide evidence that HCN2 channels possess alternating cooperative and noncooperative ligand binding behaviors (favoring two or four bound ligands rather than one or three) and are fully activated upon binding of two ligands.…”

Asymmetric perturbations of signalling oligomers

“…free vs. receptor-bound ligands). For instance, with labelled antibodies raised against each subunit Hlavackova et al, 2005) or with selective fluorescent ligands (Albizu et al, 2010), time-resolved FRET could be employed to demonstrate the asymmetric activation of various GPCR dimers at the surface of living cells and correlate local structural changes with functional states of ion channels (Kusch et al, 2012).…”

“…Hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels with pacemaker activity are primarily voltage-activated and additionally facilitated by cAMP binding at the cyclic NB IC domain. It was concluded from a confocal patch-clamp fluorometric study that zero and four cAMP (thus symmetric) bindings result in greater stability of homotetrameric HCN2 channels than one-and three-liganded channels (Kusch et al, 2012). It should be noted that for cyclic homotetramers and dimers of dimers, symmetry mismatch can lead to lower-degree symmetry.…”