Subunit-specific conductance of single homomeric and heteromeric HCN pacemaker channels at femtosiemens resolution.

In mammals, the four subunit isoforms HCN1-4 assemble to form functional homotetrameric and heterotetrameric hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels. Despite the outstanding relevance of HCN channels for organisms, including generating electrical rhythmicity in cardiac pacemaker cells and diverse types of brain neurons, key channel properties are still elusive. In particular, the unitary conductance, of HCN channels is highly controversial. We analyzed the unitary conductance at femtosiemens resolution of all four homotetrameric channels of the mouse, mHCN1-4. All conductance values are in the range of 1 pS which is exceptionally small compared to most other ion channels. Surprisingly, the conductance among the isoforms differs up to threefold (γ = 1.54 pS > γ = 0.84 pS > γ = 0.54 pS ≈ γ = 0.51 pS) though the residues in the two narrow parts of the pore, the selectivity filter and the inner gate, are conserved. Mutagenesis and all-atom molecular dynamics simulations demonstrate that the differences in the conductance are generated by different amounts of negative charges in the outer channel vestibule, which control ion accumulation. In line with these results, heterotetrameric channels exhibit intermediate unitary conductance values with respect to the homotetrameric channels. Our approach demonstrates how HCN channels can be functionally differentiated at the single-channel level, paving the way to target specific channels with selective drugs.