Publications


Structure and assembly of calcium homeostasis modulator proteins.

Nat Struct Mol Biol 27 (2020) 150-159

JL Syrjanen, K Michalski, T-H Chou, T Grant, S Rao, N Simorowski, SJ Tucker, N Grigorieff, H Furukawa

The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.


Publisher Correction: Structure and assembly of calcium homeostasis modulator proteins.

Nature structural & molecular biology 27 (2020) 305-

JL Syrjanen, K Michalski, T-H Chou, T Grant, S Rao, N Simorowski, SJ Tucker, N Grigorieff, H Furukawa

An amendment to this paper has been published and can be accessed via a link at the top of the paper.


Selectivity filter instability dominates the low intrinsic activity of the TWIK-1 K2P K+ Channel.

The Journal of biological chemistry (2019)

E Nematian-Ardestani, MF Abd-Wahab, FC Chatelain, H Sun, M Schewe, T Baukrowitz, SJ Tucker

Two-pore domain (K2P) K+ channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by post-translational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these various potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating the underlying mechanism, we observed that the low activity of the SF gate appears to arise from the inefficiency of K+ in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb+, NH4+, and Cs+, strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via a SF-mediated gating mechanism, but we found here that only very strong, non-physiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb+ currents are potently inhibited by intracellular K+ (IC50 = 2.8 mM). We conclude that TWIK-1 displays unique SF gating properties among 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.


Induced Polarization in MD Simulations of the 5HT3 Receptor Channel

(2020)

G Klesse, S Rao, S Tucker, MSP Sansom

Abstract Ion channel proteins form water-filled nanoscale pores within lipid bilayers and their properties are dependent on the complex behavior of water in a nano-confined environment. Using the pore of the 5HT3 receptor (5HT3R) we compare additive with polarizable models in describing the behavior of water in nanopores. Molecular Dynamics simulations were performed with four conformations of the channel: two closed state structures, an intermediate state, and an open state, each embedded in a phosphatidylcholine bilayer. Water density profiles revealed that for all water models, the closed and intermediate states exhibited strong dewetting within the central hydrophobic gate region of the pore. However, the open state conformation exhibited varying degrees of hydration, ranging from partial wetting for the TIP4P/2005 water model, to complete wetting for the polarizable AMOEBA14 model. Water dipole moments calculated using polarizable force fields also revealed that water molecules remaining within dewetted sections of the pore resemble gas phase water. Free energy profiles for Na+ and for Cl− ions within the open state pore revealed more rugged energy landscapes using polarizable force fields, and the hydration number profiles of these ions were also sensitive to induced polarization resulting in a substantive reduction of the number of waters within the first hydration shell of Cl− whilst it permeates the pore. These results demonstrate that induced polarization can influence the complex behavior of water and ions within nanoscale pores and provides important new insights into their chemical properties. ToC Graphic <jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="971853v1_ufig1" position="float" orientation="portrait" />


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 National Academy of Sciences (2019)

P Stansfeld, S Rao, K Klesse, S Tucker, M 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 American Association for the Advancement of Science 363 (2019) 875-880

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

<jats:p>Potassium (K<jats:sup>+</jats:sup>) 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<jats:sup>+</jats:sup> channels gated at their selectivity filter (SF), including many two-pore domain K<jats:sup>+</jats:sup> (K<jats:sub>2P</jats:sub>) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca<jats:sup>2+</jats:sup>)–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<jats:sup>+</jats:sup> occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K<jats:sup>+</jats:sup> channel activators and highlight a filter gating machinery that is conserved across different families of K<jats:sup>+</jats:sup> channels with implications for rational drug design.</jats:p>


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

BIOPHYSICAL JOURNAL 116 (2019) 301A-302A

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

BIOPHYSICAL JOURNAL 116 (2019) 248A-249A

LJ Conrad, SJ Tucker


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

BIOPHYSICAL JOURNAL 116 (2019) 398A-399A

M Arcangeletti, SJ Tucker


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

BIOPHYSICAL JOURNAL 116 (2019) 241A-241A

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 regulation of ion channel and transporter function requires the modulation of energetic barriers or ‘gates’ within their transmembrane pathways. However, despite the ever-increasing number of available structures, our understanding of these barriers is often simply determined from calculating the physical dimensions of the pore. Such approaches (e.g. the HOLE program) have worked very well in the past, but there is now considerable evidence that the unusual behaviour of water within the narrow hydrophobic spaces found within many ion channel pores can also produce energetic barriers to ion conduction without requiring physical occlusion of the permeation pathway. Several different classes of ion channels have now been shown to exploit this principle of ‘hydrophobic gating’ to regulate ion flow. However, measurement of pore radius alone is unable to identify such barriers and new tools are required for more accurate functional annotation of an exponentially increasing number of ion channel structures. We have previously shown how molecular dynamics simulations of water behaviour can be used as a proxy to accurately predict hydrophobic gates. Here we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology to predict the conductive status of new ion channel structures.


Structure and assembly of calcium homeostasis modulator proteins

(2019)

J Syrjanen, K Michalski, T-H Chou, T Grant, S Rao, N Simorowski, S Tucker, N Grigorieff, H Furukawa

Abstract Biological membranes of many tissues and organs contain large-pore channels designed to permeate a wide variety of ions and metabolites. Examples include connexin, innexin, and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), which permeate ions and ATP in a voltage-dependent manner to control neuronal excitability, taste signaling, and pathologies of depression and Alzheimer’s disease. Despite such critical biological roles, the structures and patterns of oligomeric assembly remain unclear. Here, we reveal the first structures of two CALHMs, CALHM1 and CALHM2, by single particle cryo-electron microscopy, which show novel assembly of the four transmembrane helices into channels of 8-mers and 11-mers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore-size, lipid accommodation, and channel activity.


Selectivity filter instability dominates the low intrinsic activity of the TWIK-1 K2P K+ Channel

(2019)

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

ABSTRACT Two-pore domain (K2P) K + channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/ KCNK1 ) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by post-translational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these various potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating the underlying mechanism, we observed that the low activity of the SF gate appears to arise from the inefficiency of K + in stabilizing an active ( i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb + , NH 4 + , and Cs + , strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via a SF-mediated gating mechanism, but we found here that only very strong, non-physiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb + currents are potently inhibited by intracellular K + (IC 50 = 2.8 mM). We conclude that TWIK-1 displays unique SF gating properties among 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.


A Heuristic Derived from Analysis of the Ion Channel Structural Proteome Permits the Rapid Identification of Hydrophobic Gates

(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 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 an additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavour liquid-phase water, leading to local de-wetting 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 de-wetting 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 may also facilitate the design of novel nanopores controlled by hydrophobic gates. Significance statement Ion channels are nanoscale protein pores in cell membranes. An exponentially increasing number of structures for channels means that computational methods for predicting their functional state are needed. Hydrophobic gates in ion channels result in local de-wetting of pores which functionally closes them to water and ion permeation. We use simulations of water behaviour within nearly 200 different ion channel structures to explore how the radius and hydrophobicity of pores determine their hydration vs. de-wetting behaviour. Machine learning-assisted analysis of these simulations enables us to propose a simple model for this relationship. This allows us to present an easy method for the rapid prediction of the functional state of new channel structures as they emerge.


Water and hydrophobic gates in ion channels and nanopores

Faraday Discussions Royal Society of Chemistry 209 (2018) 231-247

S Rao, SJ Tucker, G Klesse, PJ Stansfeld, GE Oakley, M Sansom, C Lynch

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

BIOPHYSICAL JOURNAL 114 (2018) 134A-134A

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


Hydrophobic Gating: Examination of Recent Ion Channel Structures

BIOPHYSICAL JOURNAL 114 (2018) 134A-134A

S Rao, G Klesse, SJ Tucker, MSP Sansom


Rare Nav1.7 variants associated with painful diabetic peripheral neuropathy

PAIN Lippincott, Williams and Wilkins 159 (2017) 469–480-

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

Diabetic peripheral neuropathy (DPN) is a common disabling complication of diabetes. Almost half of DPN patients develop neuropathic pain 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 neuropathic pain in a deeply phenotyped cohort of patients with DPN. While no rare variants were found in 78 participants with painless DPN, we identified twelve rare Nav1.7 variants in ten (out of 111) study participants with painful DPN. Five of these variants had previously been described in the context of other neuropathic pain disorders and seven have not previously been linked to neuropathic pain. Those patients with rare variants reported more severe pain and greater sensitivity to pressure stimuli on quantitative sensory testing. Electrophysiological characterization of two of the novel variants (M1852T and T1596I) demonstrated gain of function changes as a consequence of markedly impaired channel fast inactivation. By using a structural model of Nav1.7 we were also able provide further insight into the structural mechanisms underlying fast activation and the role of the C-terminal domain in this process. Our observations suggest that rare Nav1.7 variants contribute to the development neuropathic pain in patients with diabetic peripheral neuropathy. Their identification should aid understanding of sensory phenotype, patient stratification and help target treatments effectively.


Asymmetric mechanosensitivity in a eukaryotic ion channel

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

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

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.

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