C-linker residues critical for isoform-specific modulation of HCN2 by cAMP. Top: Alignment of the C-linker of HCN1 and HCN2 illustrating in red the residues that were found to have effects on the response of HCN2 to cAMP. The remaining residues that differ between isoforms are shown in blue. (A–G) Normalized conductance–voltage curves in the presence and absence of cAMP obtained by measuring tail currents of WT HCN2 and various C-linker substitutions with equivalent HCN1 residues. Mutations that significantly decreased the cAMP-dependent ΔV_1/2 are labeled red. (H) Summary of the shifts in the midpoints of activation (ΔV_1/2) induced by cAMP for the mutants shown in A–G. *, P = 0.02; **, P < 0.00003 versus WT HCN2. The numbers of patches recorded per mutant were HCN2 (n = 19), D489E G493S (n = 21), D489E G497D G493S (n = 21), K534R (n = 18), M485I K534R (n = 14), S514T K534R (n = 19), and M485I G497D S514T (n = 18). Data presented are mean ± SEM.