Subunit-specific conductance of single HCN pacemaker channels at femtosiemens resolution

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that generate rhythmic electrical activity in neuronal and cardiac pacemaker cells¹. The channels are activated by hyperpolarisation of the membrane voltage and additionally tuned by the second messenger cAMP at sympathetic stimulation. There are four mammalian isoforms, HCN1-4^2–4. The single-channel conductance,γ, of HCN channels remains debated, with conflicting results ranging from near 1.5 pS for HCN2^5,6to tens of pS for HCN1⁷, HCN2⁷and HCN4^7,8, though the pore structure, viewed to determine the conductance^9,10, is either identical or highly conserved. To resolve this controversy, we analyzed all four mouse isoforms mHCN1-4 at femtosiemens resolution. We show that mHCN1, mHCN3 and mHCN4 also generate small conductance values, even smaller than that of mHCN2 with the sequenceγ_mHCN2=1.54 pS >γ_mHCN1=0.84 pS >γ_mHCN3=0.54 pS ≍γ_mHCN4=0.51 pS. As shown by systematic mutagenesis and molecular dynamic simulations, the differences in the conductance are neither generated by the selectivity filter nor the inner gate⁹, but by defined negative charges in the outer channel vestibule increasing cation occupation. In line with these results, heteromers of mHCN2 with either mHCN1, mHCN3 or mHCN4 lead to graded single-channel currents in-between those of the respective homomeric channels. Our approach at femtosiemens resolution provides insight into the function of recombinant and native HCN channels at the level of single subunits and is thus promising for the development of subunit-specific drugs acting on these clinically highly relevant channels¹¹.