Alanine scanning of residues near the intracellular end of S6. (A) Alignment of primary sequences from mHCN2, MthK, KcsA, and Kv1.2 channels. Selectivity filter and S6 segment are indicated by red squares. Numbers on the top refer to the residue positions in mHCN2. Asterisks indicate the sequence number for the mHCN2 channel. Underlining indicates the region scanned by alanine replacement. Green and magenta letters indicate that the corresponding alanine replacements either increase (green) or decrease (magenta) the dynamic cAMP binding. (B) Normalized macroscopic current traces for WT (black), mHCN2/I432A (magenta), and mHCN2/H434A (green) channels. Voltage step and timing of fluorescent image collection are shown on top of the current traces. (C) Representative fluorescence images for I432A and H434A mutant channels. Timing of fluorescence image collection is shown in B. (D) Profiles of cAMP binding in response to a hyperpolarizing voltage step for WT mHCN2 (black), mHCN2/I432A (magenta), and mHCN2/H434A (green) channels. Fluorescence intensities are normalized to the value collected at −40 mV. (E) Summary of the PCF results. The mHCN2 channel contains two endogenous alanine residues in this region, A429 and A435. A435 was tested by a glycine replacement. A429 was not tested so the result represents the WT channel value. Independent samples t test shows that each of the following alanine mutants, T426A, M430A, F431A, I432A, and H434A, are significantly different from WT (A429; P < 0.01). (D and E) Error bars indicate standard error. (F) Structure model of the S5-pore-S6 region from the mHCN2 channel. The crystal structure of Kv1.2/2.1 chimera (Protein Data Bank accession no. 2R9R; Long et al., 2007) was used as the template for modeling. Side chains of T426, M430, and H434, of which the corresponding alanine replacement mutations increased cAMP binding, are shown in green. Side chains of F431 and I432, of which the alanine replacement mutations reduced cAMP binding, are shown in magenta.