Publications


A heuristic derived from analysis of the ion channel structural proteome permits the rapid identification of hydrophobic gates.

Proceedings of the National Academy of Sciences of the United States of America 116 (2019) 13989-13995

S Rao, G Klesse, PJ Stansfeld, SJ Tucker, MSP Sansom

Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An exponentially increasing number of channel structures captured in different conformational states are now being determined; however, these newly resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore, and it is now known that more accurate annotation of their conductive state requires additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavor liquid-phase water, leading to local dewetting, which will form an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore dewetting by applying molecular dynamics simulations and machine learning to nearly 200 ion channel structures. This allows us to propose a simple simulation-free heuristic model that rapidly and accurately predicts the presence of hydrophobic gates. This not only enables the functional annotation of new channel structures as soon as they are determined, but also may facilitate the design of novel nanopores controlled by hydrophobic gates.


CHAP: A Versatile Tool for the Structural and Functional Annotation of Ion Channel Pores.

Journal of molecular biology (2019)

G Klesse, S Rao, MSP Sansom, SJ Tucker

The control of ion channel permeation requires the modulation of energetic barriers or "gates" within their pores. However, such barriers are often simply identified from the physical dimensions of the pore. Such approaches have worked well in the past,but there is now evidence that the unusual behaviour of water within narrow hydrophobic pores can produce an energetic barrier to permeation without requiring steric occlusion of the pathway. Many different ion channels have now been shown to exploit "hydrophobic gating" to regulate ion flow, and it is clear that new tools are required for more accurate functional annotation of the increasing number of ion channel structures becoming available. We have previously shown how molecular dynamics simulations of water can be used as a proxy to predict hydrophobic gates, and we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology.


A pharmacological master key mechanism that unlocks the selectivity filter gate in K+ channels.

Science (New York, N.Y.) 363 (2019) 875-880

M Schewe, H Sun, Ü Mert, A Mackenzie, ACW Pike, F Schulz, C Constantin, KS Vowinkel, LJ Conrad, AK Kiper, W Gonzalez, M Musinszki, M Tegtmeier, DC Pryde, H Belabed, M Nazare, BL de Groot, N Decher, B Fakler, EP Carpenter, SJ Tucker, T Baukrowitz

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.


A Pharmacological Masterkey Mechanism to Unlock the Selectivity Filter Gate in K plus Channels

(2019)

M Schewe, H Sun, A Mackenzie, ACW Pike, F Schulz, C Constantin, AK Kiper, LJ Conrad, W Gonzalez, BL de Groot, N Decher, B Fakler, EP Carpenter, SJ Tucker, T Baukrowitz


Insights into Selectivity Filter Gating of K2P Channels from Single Channel Recordings

(2019)

LJ Conrad, SJ Tucker


Systematic Scanning Mutagenesis of the Pore Helices in the TREK-2 K2P Channel

(2019)

M Arcangeletti, SJ Tucker


Functional Annotation of Ion Channel Structures: Predicting Pore Solvation States Based on Local Radius and Hydrophobicity

(2019)

S Rao, G Klesse, PJ Stansfeld, SJ Tucker, MSP Sansom


CHAP: A versatile tool for the structural and functional annotation of ion channel pores

(2019)

G Klesse, S Rao, MSP Sansom, S Tucker

Abstract The control of ion channel permeation requires the modulation of energetic barriers or ‘gates’ within their pores. However, such barriers are often simply identified from the physical dimensions of the pore. Such approaches have worked well in the past, but there is now evidence that the unusual behaviour of water within narrow hydrophobic pores can produce an energetic barrier to permeation without requiring steric occlusion of the pathway. Many different ion channels have now been shown to exploit ‘hydrophobic gating’ to regulate ion flow, and it is clear that new tools are required for more accurate functional annotation of the increasing number of ion channel structures becoming available. We have previously shown how molecular dynamics simulations of water can be used as a proxy to predict hydrophobic gates, and we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology.


Selectivity Filter Instability Dominates the Low Intrinsic Activity of the TWIK-1 K2P K+ Channel

(2019)

E Nemetian-Ardestani, F Abd-Wahab, F Chatelain, H Sun, M Schewe, T Baukrowitz, S Tucker

ABSTRACT The functional properties of the TWIK-1 ( KCNK1 ) Two-Pore Domain (K2P) K + channel remain poorly characterized due to the very low levels of functional activity it produces when heterologously expressed. Several underlying reasons have been proposed including retention in intracellular organelles, inhibition by post-translational sumoylation, a hydrophobic barrier within the pore, and a low intrinsic open-probability of the selectivity filter (SF) gate. By evaluating these different mechanisms, we found the latter to dominate this low intrinsic functional activity and investigated the underlying mechanism. The low activity of the SF gate appears to result from the inefficiency of K + in stabilizing an active (i.e. conductive) SF conformation, while other permeant ion species such as Rb + , NH 4 + and Cs + strongly promote a pH-dependent activated conformation. Furthermore, while many K2P channels are activated by membrane depolarization via a SF-mediated gating mechanism, only very strong, non-physiological depolarization produces voltage-dependent activation and the channel displays unusual inactivation kinetics. Remarkably, we observed that TWIK-1 Rb + currents were potently inhibited by intracellular K + (IC 50 = 2.8 mM). TWIK-1 therefore displays unique SF gating properties amongst the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K + appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface.


Identification of Hydrophobic Gates by Simulation-Enabled Analysis of the Ion Channel Structural Proteome

(2018)

S Rao, G Klesse, P Stansfeld, S Tucker, MSP Sansom

Abstract Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An increasing number of channel structures captured in different conformational states are being determined as a result of advances in membrane protein structural biology. However, newly resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore and it is now known that more accurate annotation of their conductive state requires an additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavour liquid-phase water, leading to local de-wetting and an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore de-wetting by applying molecular dynamics-based calculations to nearly 200 ion channel structures. Using machine learning, we propose a simple heuristic model that allows the rapid prediction of the state of hydrophobic gates based on analysis of both local radius and hydrophobicity in ion channel structures and nanopores.


Water and hydrophobic gates in ion channels and nanopores.

Faraday discussions 209 (2018) 231-247

S Rao, CI Lynch, G Klesse, GE Oakley, PJ Stansfeld, SJ Tucker, MSP Sansom

Ion channel proteins form nanopores in biological membranes which allow the passage of ions and water molecules. Hydrophobic constrictions in such pores can form gates, i.e. energetic barriers to water and ion permeation. Molecular dynamics simulations of water in ion channels may be used to assess whether a hydrophobic gate is closed (i.e. impermeable to ions) or open. If there is an energetic barrier to water permeation then it is likely that a gate will also be impermeable to ions. Simulations of water behaviour have been used to probe hydrophobic gates in two recently reported ion channel structures: BEST1 and TMEM175. In each of these channels a narrow region is formed by three consecutive rings of hydrophobic sidechains and in both cases such analysis demonstrates that the crystal structures correspond to a closed state of the channel. In silico mutations of BEST1 have also been used to explore the effect of changes in the hydrophobicity of the gating constriction, demonstrating that substitution of hydrophobic sidechains with more polar sidechains results in an open gate which allows water permeation. A possible open state of the TMEM175 channel was modelled by the in silico expansion of the hydrophobic gate resulting in the wetting of the pore and free permeation of potassium ions through the channel. Finally, a preliminary study suggests that a hydrophobic gate motif can be transplanted in silico from the BEST1 channel into a simple β-barrel pore template. Overall, these results suggest that simulations of the behaviour of water in hydrophobic gates can reveal important design principles for the engineering of gates in novel biomimetic nanopores.


A Newly Available Tool for Functional Annotation of Ion Channel Structures Based on Molecular Dynamics Simulations

(2018)

G Klesse, S Rao, PJ Stansfeld, MSP Sansom, SJ Tucker


Hydrophobic Gating: Examination of Recent Ion Channel Structures

(2018)

S Rao, G Klesse, SJ Tucker, MSP Sansom


Rare NaV1.7 variants associated with painful diabetic peripheral neuropathy.

Pain 159 (2018) 469-480

I Blesneac, AC Themistocleous, C Fratter, LJ Conrad, JD Ramirez, JJ Cox, S Tesfaye, PR Shillo, ASC Rice, SJ Tucker, DLH Bennett

Diabetic peripheral neuropathy (DPN) is a common disabling complication of diabetes. Almost half of the patients with DPN develop neuropathic pain (NeuP) for which current analgesic treatments are inadequate. Understanding the role of genetic variability in the development of painful DPN is needed for improved understanding of pain pathogenesis for better patient stratification in clinical trials and to target therapy more appropriately. Here, we examined the relationship between variants in the voltage-gated sodium channel NaV1.7 and NeuP in a deeply phenotyped cohort of patients with DPN. Although no rare variants were found in 78 participants with painless DPN, we identified 12 rare NaV1.7 variants in 10 (out of 111) study participants with painful DPN. Five of these variants had previously been described in the context of other NeuP disorders and 7 have not previously been linked to NeuP. Those patients with rare variants reported more severe pain and greater sensitivity to pressure stimuli on quantitative sensory testing. Electrophysiological characterization of 2 of the novel variants (M1852T and T1596I) demonstrated that gain of function changes as a consequence of markedly impaired channel fast inactivation. Using a structural model of NaV1.7, we were also able to provide further insight into the structural mechanisms underlying fast inactivation and the role of the C-terminal domain in this process. Our observations suggest that rare NaV1.7 variants contribute to the development NeuP in patients with DPN. Their identification should aid understanding of sensory phenotype, patient stratification, and help target treatments effectively.


The effects of stretch activation on ionic selectivity of the TREK-2 K2P K+ channel.

Channels (Austin, Tex.) 11 (2017) 482-486

E Nematian-Ardestani, V Jarerattanachat, P Aryal, MSP Sansom, SJ Tucker

The TREK-2 (KCNK10) K2P potassium channel can be regulated by variety of polymodal stimuli including pressure. In a recent study, we demonstrated that this mechanosensitive K+ channel responds to changes in membrane tension by undergoing a major structural change from its 'down' state to the more expanded 'up' state conformation. These changes are mostly restricted to the lower part of the protein within the bilayer, but are allosterically coupled to the primary gating mechanism located within the selectivity filter. However, any such structural changes within the filter also have the potential to alter ionic selectivity and there are reports that some K2Ps, including TREK channels, exhibit a dynamic ionic selectivity. In this addendum to our previous study we have therefore examined whether the selectivity of TREK-2 is altered by stretch activation. Our results reveal that the filter remains stable and highly selective for K+ over Na+ during stretch activation, and that permeability to a range of other cations (Rb+, Cs+ and NH4+) also does not change. The asymmetric structural changes that occur during stretch activation therefore allow the channel to respond to changes in membrane tension without a loss of K+ selectivity.


Dynamic role of the tether helix in PIP2-dependent gating of a G protein-gated potassium channel.

The Journal of general physiology (2017)

E Lacin, P Aryal, IW Glaaser, K Bodhinathan, E Tsai, N Marsh, SJ Tucker, MSP Sansom, PA Slesinger

G protein-gated inwardly rectifying potassium (GIRK) channels control neuronal excitability in the brain and are implicated in several different neurological diseases. The anionic phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is an essential cofactor for GIRK channel gating, but the precise mechanism by which PIP2 opens GIRK channels remains poorly understood. Previous structural studies have revealed several highly conserved, positively charged residues in the "tether helix" (C-linker) that interact with the negatively charged PIP2 However, these crystal structures of neuronal GIRK channels in complex with PIP2 provide only snapshots of PIP2's interaction with the channel and thus lack details about the gating transitions triggered by PIP2 binding. Here, our functional studies reveal that one of these conserved basic residues in GIRK2, Lys200 (6'K), supports a complex and dynamic interaction with PIP2 When Lys200 is mutated to an uncharged amino acid, it activates the channel by enhancing the interaction with PIP2 Atomistic molecular dynamic simulations of neuronal GIRK2 with the same 6' substitution reveal an open GIRK2 channel with PIP2 molecules adopting novel positions. This dynamic interaction with PIP2 may explain the intrinsic low open probability of GIRK channels and the mechanism underlying activation by G protein Gβγ subunits and ethanol.


Asymmetric mechanosensitivity in a eukaryotic ion channel.

Proceedings of the National Academy of Sciences of the United States of America 114 (2017) E8343-E8351

MV Clausen, V Jarerattanachat, EP Carpenter, MSP Sansom, SJ Tucker

Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian two-pore domain (K2P) K+ channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli.


Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel.

Structure (London, England : 1993) 25 (2017) 708-718.e2

P Aryal, V Jarerattanachat, MV Clausen, M Schewe, C McClenaghan, L Argent, LJ Conrad, YY Dong, ACW Pike, EP Carpenter, T Baukrowitz, MSP Sansom, SJ Tucker

The mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the "down" to "up" conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.


A BEST example of channel structure annotation by molecular simulation.

Channels (Austin, Tex.) 11 (2017) 347-353

S Rao, G Klesse, PJ Stansfeld, SJ Tucker, MSP Sansom

An increasing number of ion channel structures are being determined. This generates a need for computational tools to enable functional annotation of channel structures. However, several studies of ion channel and model pores have indicated that the physical dimensions of a pore are not always a reliable indicator of its conductive status. This is due to the unusual behavior of water within nano-confined spaces, resulting in a phenomenon referred to as "hydrophobic gating". We have recently demonstrated how simulating the behavior of water within an ion channel pore can be used to predict its conductive status. In this addendum to our study, we apply this method to compare the recently solved structure of a mutant of the bestrophin chloride channel BEST1 with that of the wild-type channel. Our results support the hypothesis of a hydrophobic gate within the narrow neck of BEST1. This provides further validation that this simulation approach provides the basis for an accurate and computationally efficient tool for the functional annotation of ion channel structures.


Regulation of Two-pore Domain K plus Channels by Natural Effectors and Pharmacological Agents

(2017)

M Schewe, F Schulz, U Mert, H Sun, H Belabed, M Musinszki, T Koehler, M Tegtmeier, M Nazare, EP Carpenter, SJ Tucker, T Baukrowitz

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