cAMP can bind to the CNBD of HCN4 in CHO cells. (A) Fluorescence emission spectra for binding of 3 µM 8-NBD-cAMP to GST fusion proteins. GST, unconjugated glutathione S-transferase; HCN4-C2, residues 917–1201 of the distal C terminus of HCN4 (distal to the CNBD); HCN4-CNBD, residues 528–737 of the C terminus of HCN4, which include the C-linker and CNBD; HCN2-CNBD, residues 443–645 of HCN2, which include the C-linker and CNBD. Spectra are representative of three independent trials. (B) Deactivation time constants for HCN4 in excised patches from CHO cells before (black) and after (red) perfusion with 100 µM cAMP. Asterisk indicates statistical significance compared with control (large symbols; P = 0.009; paired t test). Small symbols represent data from individual patches, and lines connect time constants acquired in the same patches. (Inset) Representative tail currents at −50 mV after voltage steps to −150 mV for a patch expressing HCN4 before (black) and after (red) perfusion of 100 µM cAMP.