Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate inward cation currents that contribute to rhythmic electrical activity in the heart and brain. This thesis investigates the gating mechanisms of HCN channels, focusing on voltage-dependent activation and intracellular Mg²⁺ block. Using substituted cysteine accessibility and electrophysiological analysis, the S4 transmembrane segment of HCN1 is shown to function as a conserved voltage sensor that undergoes state-dependent movement, despite reversed gating relative to Kv channels. Deletion analyses indicate that differences in cyclic AMP sensitivity among HCN isoforms arise from their C-terminal regions. Additionally, intracellular Mg²⁺ acts as a voltage-dependent pore blocker, reducing outward currents and contributing to inward rectification. Together, these findings reveal complementary intrinsic and extrinsic mechanisms regulating HCN channel gating.
