Role Of Interactions Between Transmembrane And C-Terminal Regions In Voltage-Dependent Activation Of Hcn4 Channels

The HCN4 subtype of Hyperpolarization- and Cyclic Nucleotide-activated (HCN) channels is noted for slower kinetics and a hyperpolarized activation midpoint voltage (V1/2) compared to the HCN2 subtype. To test whether the HCN4 transmembrane (TM) region requires interactions with the C-terminal region to determine V1/2, we substituted the HCN4 TM region into previously characterized HCN2 channels with various truncations of the C-terminal region. Substituting the HCN4 TM region into HCN2 channels - either full-length or truncated after the cyclic nucleotide-binding (CNB) fold - resulted in a significant (more than 8 mV) negative shift of V1/2. Strikingly, in HCN2 channels lacking the CNB fold, or channels where the majority of the CNB fold was present except for the final C-helix, the HCN4 TM substitution failed to shift V1/2 significantly (less than 2 mV difference). This suggests that the hyperpolarized V1/2 of HCN4 channels relative to other HCN subtypes relies on a functional interaction of the TM and C-terminal regions, and that the specific conformation supporting this inhibitory interaction requires an intact CNB fold. To test whether this inhibitory interaction coincides with any inhibition caused by eliminating CNB fold-mediated cAMP potentiation, we repeated the HCN4 TM substitution in a full length HCN2 channel whose CNB fold is mutated (R591E) so it cannot bind cAMP. HCN2 R591E exhibits V1/2 20 mV more negative than cAMP-potentiated HCN2 channels, yet the HCN4 TM substitution in HCN2 R591E produced a further 10 mV negative V1/2 shift, matching that produced by HCN4 TM substitution in cAMP-potentiated channels. This suggests a new role for the CNB fold in supporting an inhibitory interaction between the C-terminal region and the HCN4 TM region, independent of and additive with the well-known autoinhibition caused by eliminating cAMP potentiation.