Structural basis for activation of voltage sensor domains in an ion channel TPC1

Abstract

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca-binding site (Ca), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca to a cytoplasmic site (Ca). An X-ray structure with Ca removed and a near-atomic resolution cryo-EM structure with Ca removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca ions for activation.

Helix breaking transition in the S4 of HCN channel is critical for hyperpolarization-dependent gating

“…The mechanism of voltage-sensor activation has been a focus of many studies (Larsson et al, 1996, Chanda et al, 2005; Posson et al, 2005; Long et al, 2005; Swartz, 2008; Delemotte et al, 2011; Amaral et al, 2012; Henrion et al, 2012; Jensen et al, 2012; Kintzer et al, 2018; Li et al, 2015; Bezanilla, 2018; Wisedchaisri et al, 2019) over the past two decades, and the emerging consensus in the field is that VSD activation involves a helical screw motion across the membrane relative to the surrounding S1-S3 bundle which acts as a gating scaffold. In all these channels, the measured gating charge originates from the physical displacement of S4 in a relatively static electrostatic environment which is also conserved across different voltage sensor domains (Souza et al, 2014).…”

Parallel adaptation in autopolyploid Arabidopsis arenosa is dominated by repeated recruitment of shared alleles

“…To test this, we performed structural homology modelling of all alleles found in A. arenosa (Fig 3d-g), using two crystallographically-determined structures as a template (PDB codes 5DQQ and 5E1J 46,47 ). In the tertiary structure, residue 630 sits adjacent to the Asn residue (Asn627 in A. arenosa), which forms the pore's constriction point and has been shown to control ion selectivity in A. thaliana [46][47][48][49] . In A. thaliana this Asn627 residue, when substituted by sitedirected mutagenesis to the human homolog state can cause Na+ non-selective A. thaliana TPC1 to adopt the Na+ selectivity of human TPC1 47 .…”

The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

“…The tilting motion of S4 is also consistent with previous cysteine accessibility experiments in HCN channels 24,25 . Furthermore, the rearrangement is similar to the S4 movement in VSD2 of the depolarization-activated two-pore channel 1 (TPC1) inferred from structures of mouse TPC1 (up state) and Ba 2+ -inhibited Arabidopsis TPC1 (down state) at 0 mV 26,27 (Supplementary Video 6), but is incompatible with other models proposing that the S4 helix is relatively immobile 28 . Moreover, considering the tight packing of S4 and S5 helices in HCN channels 11 , the downward and tilting movement of the S4 helix could bypass the short S4-S5 linker to influence S5 directly, thus allowing the channels to open 29,30 .…”