Alkali metal cations modulate the geometry of different binding sites in HCN4 selectivity filter for permeation or block.

Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels are important for timing biological processes like heartbeat and neuronal firing. Their weak cation selectivity is determined by a filter domain with only two binding sites for K+ and one for Na+. The latter acts as a weak blocker, which is released in combination with a dynamic widening of the filter by K+ ions, giving rise to a mixed K+/Na+ current. Here, we apply molecular dynamics simulations to systematically investigate the interactions of five alkali metal cations with the filter of the open HCN4 pore. Simulations recapitulate experimental data like a low Li+ permeability, considerable Rb+ conductance, a block by Cs+ as well as a punch through of Cs+ ions at high negative voltages. Differential binding of the cation species in specific filter sites is associated with structural adaptations of filter residues. This gives rise to ion coordination by a cation-characteristic number of oxygen atoms from the filter backbone and solvent. This ion/protein interplay prevents Li+, but not Na+, from entry into and further passage through the filter. The site equivalent to S3 in K+ channels emerges as a preferential binding and presumably blocking site for Cs+. Collectively, the data suggest that the weak cation selectivity of HCN channels and their block by Cs+ are determined by restrained cation-generated rearrangements of flexible filter residues.

Detection of ligand binding to purified HCN channels using fluorescence-based size exclusion chromatography.

Biochemical measurements of ligand binding to eukaryotic membrane proteins are challenging because they can require large amounts of purified protein. For this reason, ligand binding is preferentially evaluated on soluble domains rather than on the full length proteins. In this chapter, we describe the use of fluorescence size exclusion chromatography-based thermostability (FSEC-TS) as an assay to monitor ligand binding to the full length mammalian ion channel HCN4. FSEC-TS monitors the effect of the ligand on the thermal denaturation curve of the protein by following the fluorescence of a fused GFP protein. Changes in the melting temperature (Tm) provide a quantitative value for measuring ligand-protein interaction. As a proof of concept, we describe here the protocol for monitoring the binding of the second messenger cAMP and of the known HCN drug Ivabradine to the purified GFP-HCN4 channel. cTMP, a known non-binder of HCN channels, is used as a control. Due to the small amount of protein required, the assay represents a high-throughput screening system for evaluating binding of small molecules to full length proteins.

Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.