The hyperpolarization-activated cyclic nucleotide-gated ion channel 4 (HCN4) current increases due to cAMP binding and is well-recognized to contribute to adrenergically driven heart rate acceleration. HCN4 current also increases with heat by an unknown mechanism(s). We use thermodynamical and homology computational modeling, site-directed mutagenesis, and mouse models to identify a concise motif on the S4-S5 linker of HCN4 channels (M407/Y409) that determines HCN4 current (If) responses to heat. This motif is required for heat-triggered rate acceleration in cardiac pacemaker cells, isolated hearts and in vivo. Surprisingly, a loss of function M407/Y409 motif mutation prevented not only normal heat but also cAMP responses, suggesting that the heat-sensing machinery within the S4-S5 linker is essential for operating the cAMP allosteric pathway and is central to HCN4 gating modulation. The M407/Y409 motif is conserved across all HCN family members suggesting that HCN channels participate broadly in coupling heat to changes in cell membrane excitability.
