S4–S5 linker movement during activation and inactivation in voltage-gated K ⁺ channels

Abstract

The S4-S5 linker physically links voltage sensor and pore domain in voltage-gated ion channels and is essential for electromechanical coupling between both domains. Little dynamic information is available on the movement of the cytosolic S4-S5 linker due to lack of a direct electrical or optical readout. To understand the movements of the gating machinery during activation and inactivation, we incorporated fluorescent unnatural amino acids at four positions along the linker of the Shaker K channel. Using two-color voltage-clamp fluorometry, we compared S4-S5 linker movements with charge displacement, S4 movement, and pore opening. We found that the proximal S4-S5 linker moves with the S4 helix throughout the gating process, whereas the distal portion undergoes a separate motion related to late gating transitions. Both pore and S4-S5 linker undergo rearrangements during C-type inactivation. In presence of accelerated C-type inactivation, the energetic coupling between movement of the distal S4-S5 linker and pore opening disappears.

Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels

“…The VSD and the PD are connected by the S4-S5 linker, which, in the canonical model of voltage-dependent coupling, acts as the molecular conduit underlying electromechanical coupling between these two domains ( Fig. 1B) (10,11,(20)(21)(22)(23)(24). This model supposes that movement of each S4 pulls on the S4-S5 linker, which in turn opens a single gate in the pore.…”

“…the S4-S5 linker and the S6 to open the pore gate (10,11,(20)(21)(22)(23)(24). However, HCN channels are opened by hyperpolarizing voltage steps.…”

Two-stage electro-mechanical coupling of a KV channel in voltage-dependent activation

“…K V channels contain the voltage-sensor domains (VSDs) formed by the transmembrane segments S1-S4 and the pore domain (PD) formed by the segments S5-S6 1,2 . The VSD activates in a stepwise manner from the resting to the intermediate state before finally arriving at the activated state 3-14 (see also attached unpublished manuscript), which triggers pore opening via electro-mechanical (E-M) coupling [15][16][17][18][19] . The primary structural determinant underlying the E-M coupling process has classically been assigned to the linking helix between the VSD and the PD (S4-S5 linker or S4-S5L), with the physical movement of the VSD exerting an allosteric "tug" on key elements of the PD to induce pore opening [15][16][17][18][19][20] .…”

The EAG Voltage-Dependent K+ Channel Subfamily: Similarities and Differences in Structural Organization and Gating

“…This is typical of the Kv1 to Kv9 (Shaker-type) subfamilies, but also occurs in the Nav and Cav voltage-gated sodium and calcium channels (Wisedchaisri et al, 2019), and in the channels from the transient receptor (TRP) family, which are structurally related to Kv channels but are generally non-selective among cations and in most cases voltage-independent (reviewed in Barros et al, 2019). There are indications that in the domain-swapped and voltage-dependent type of channels, an electromechanical coupling between the VSD and the gate exists, in which the S4-S5 linker, acting as a mechanical lever, transmits to the cytoplasmic gate the force generated by the VSD conformational rearrangements, leading to channel activation and/or deactivation (Long et al, 2005;Labro et al, 2008;Vardanyan and Pongs, 2012;Blunck and Batulan, 2012;Jensen et al, 2012;Chowdury et al, 2014;Kalstrup and Blunck, 2018).…”

“…Note also that in many of the non-domain swapped channels gating is controlled by the binding of diverse ligands to intracellular regions that show an extensive domain swapping between the tetramer subunits (Stevens et al, 2009;Gustina and Trudeau, 2011;Gianulis et al, 2013;Haitin et al, 2013;Ng et al, 2014;Whicher and MacKinnon, 2016;Wang and MacKinnon, 2017;Zhao et al, 2017;Barros et al, 2019). In any case, it is clear that in non-domain-swapped, but genuinely voltage-dependent Kv channels, such as Kv10.1 and Kv11.1, the short length and organization of the S4-S5 linker would not allow it to act as a rigid mechanical lever able to pull apart the N-terminal portion of S5 and the C-terminal end of S6 to open the cytoplasmic channel gate, as it seems to happen in the canonical voltage-gated K + channels (Long et al, 2005;Labro et al, 2008;Vardanyan and Pongs, 2012;Blunck and Batulan, 2012;Jensen et al, 2012;Chowdury et al, 2014;Kalstrup and Blunck, 2018). Indeed, pioneer studies with Kv10.1 and Kv11.1 channels in which the separate N-and C-terminal halves of the protein were expressed after breaking the covalent continuity of the S4-S5 linker (S4-S5 split channels), demonstrated the production of voltage-gated channels exhibiting voltage-sensing and permeation properties similar to those of the complete protein (Lorinczi et al, 2015).…”