Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents (5). In the present study, we show that intracellular Mg 2ϩ functions as a voltagedependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg 2ϩ is in the physiological range, with Mg 2ϩ binding with an IC50 of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at ϩ50 mV. The effective electrical distance for the Mg 2ϩ binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg 2ϩ , suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg 2ϩ acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg 2ϩ may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg 2ϩ could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential. inward rectifying; hyperpolarization-activated current; instantaneous currents; divalent block; cysteine; hyperpolarization-activated cyclic nucleotide RHYTHMIC ACTIVITY OF PACEMAKER cells in the heart and thalamic neurons in the brain are dependent on the inward I h current through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (29,36). Following an action potential in pacemaker cells, I h contributes to the currents that slowly depolarize the membrane potential to threshold, thereby initiating another action potential (7). Four mammalian HCN channels have been cloned: HCN1-HCN4 (11,21,35). The deletion of HCN channels, or the presence of naturally occurring mutations in HCN channels, has been shown to have significant physiological consequences (20,25,37,40). HCN2 knockout mice, for example, showed spontaneous absence seizures and cardiac sinus dysrhythmia (20).HCN channels are members of the superfamily of voltagegated ion channels, possessing features such as a tetrameric structure, with each subunit containing six transmembrane domains (S1-S6) (29,36). In addition, HCN channels have a pore domain that shows conservation with voltage-gated potassium (Kv) channels, including a GYG signature motif in the selectivity filter, even though HCN channels are only modestly more selective (3:1) for K ϩ over Na ϩ (11, 21). Similar to Kv channels, HCN channels have an intracellular gate at the base of S6 that prevents access of ions to the pore (32, 33)....

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