CNBD residues critical for isoform-specific modulation of HCN2 by cAMP. Top: Alignment of the CNBD 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–E) Normalized conductance-voltage curves in presence and absence of cAMP obtained by measuring tail currents of HCN2 carrying various CNBD substitutions. Labeled red are CNBD mutations in the background of the previously identified nonneutral C-linker mutations. (F) Summary of the shifts in the midpoints of activation (ΔV_1/2) induced by cAMP for the mutants shown in A–E. The background of the CNBD mutations tested (the triple mutant M485I G497D S514T) is abbreviated by a plus sign (+) in the mutant labels. *, P < 0.04; **, P < 0.00001 versus the triple mutant M485I, G497D, and S514T. The numbers of patches recorded per mutant were M485I G497D S514T (n = 18), M485I G497D S514T V562A S563G (n = 21), M485I G497D S514T L565I (n = 22), M485I G497D S514T L565I (n = 21), M485I G497D S514T S575T (n = 21), and HCN2/1 (n = 19). Data presented are mean ± SEM.