S4 Movement in a Mammalian HCN Channel

Abstract

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.

Slow Conformational Changes of the Voltage Sensor during the Mode Shift in Hyperpolarization-Activated Cyclic-Nucleotide-Gated Channels

“…A, Sequence alignment of spHCN and HCN1 S4 domains. Marked residues exhibit state-dependent modification by intracellular (*), extracellular (#), or both intracellular and extracellular reagents (&), or state-independent modification by either intracellular or extracellular reagents (o) (Männikkö et al, 2002;Bell et al, 2004;Vemana et al, 2004). B, C, Representative current (top) and fluorescence (bottom) records from Alexa-488 C5-maleimide-exposed wt spHCN channels (B) and 326C channels (C).…”

“…2 A) that showed a stateindependent accessibility for N-terminal S4 residues (residues 326 -332 in spHCN channels) (Männikkö et al, 2002;Bell et al, 2004;Vemana et al, 2004). In contrast, residues in the middle and C-terminal end of S4 (residues 333-349 in spHCN channels) are accessible in a state-dependent manner (Männikkö et al, 2002;Bell et al, 2004;Vemana et al, 2004), suggesting that these residues are the voltage-sensing charges that undergo the transmembrane movement that triggers channel opening. The mode shift itself exhibits little, if any, voltage dependence (Elinder et al, 2006), suggesting no large inward movement of the gating charges during the mode shift.…”

“…Most of the channels in this family, such as voltage-gated K ϩ and Na ϩ channels, are activated by an outward movement of positive charges in the fourth transmembrane domain (S4) in response to membrane depolarization (Hille et al, 1999). However, HCN channels open by inward movement of positive charges in S4 in response to membrane hyperpolarization (Männikkö et al, 2002;Bell et al, 2004;Vemana et al, 2004). The activation of HCN channels is also modulated by cyclic nucleotides that bind directly to the channel C terminus (Robinson and Siegelbaum, 2003).…”

Hyperpolarization-Activated Cation Channels: From Genes to Function

“…The fact that these channels are differentially sensitive to the direction of the voltage change and that the activation curves in mode I and mode II are separated by up to 50 mV is defined as "voltage hysteresis." This interesting gating behavior of spHCN is probably the result of a slow conformational change attributable to lateral movement of S4 that stabilizes the inward position of S4 upon hyperpolarization (52,53,133,245,246,405). HCN1 channels behave similarly as spHCN channels with respect to hysteresis behavior and mode shift (246).…”

Reversed voltage-dependent gating of a bacterial sodium channel with proline substitutions in the S6 transmembrane segment

“…Studies of the S4 segments of HCN channels has confirmed that they move outward upon depolarization like other voltage-gated channels (27)(28)(29), even though HCN channels are activated by hyperpolarization. These results indicate that an alteration in the coupling of outward movement of the S4 voltage sensors to eventual opening of the pore underlies hyperpolarization-activated pore opening in HCN channels.…”