Anionic lipids unlock the gates of select ion channels in the pacemaker family

Schmidpeter, Philipp A. M.; Wu, Di; Rheinberger, Jan; Riegelhaupt, Paul M.; Tang, Haiping; Robinson, Carol V.; Nimigean, Crina M.

doi:10.1038/s41594-022-00851-2

Cited by 25 publications

(25 citation statements)

References 74 publications

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“…The C-linker/CNBD of the activated, pre-open state (SthK Y26F) is identical to those in the three open states (SthK R120A). Furthermore, the conformational changes between the CNBD/C-linker domains in the closed and the activated/ open state observed in our structures, are consistent with AFM and DEER measurements of SthK as well as with structures of the closed and open states of SthK and of the eukaryotic CNG channels TAX-4 and CNGA1 14,16,23,27,34 . The fact that mutations in the VSD lead to conformational changes in the CNBDs and the pore, highlights the allosteric link between the voltage sensor domain, ligand-binding domain, and the channel pore (Fig.…”

Section: Discussionsupporting

confidence: 89%

“…3b, c; O1: 50037 particles, ~5%; O2: 149792 particles, ~14%; O3: 46629 particles, ~5%). The strain generated by elevating and rotating the C-linker/CNBD domains in SthK R120A is efficiently transmitted to the pore lining S6 helices that are rotated outwards, opening the intracellular bundle crossing region 27 . S6 hinges outwards at Ala208, located right underneath the selectivity filter (Fig.…”

Section: Sthk R120a Reveals Multiple Open Statesmentioning

confidence: 99%

“…3e). In the closed state, the bundle crossing is stabilized by an inter-subunit salt-bridge between Arg136 on S5 and Asp226 on S6 27 . In the activated, pre-open conformation, although the pore is still closed, this salt-bridge is broken (Fig.…”

Section: Vsd Positioning Allows For Different Gate Expansionsmentioning

confidence: 99%

“…Cyclic nucleotides are very efficient in opening eukaryotic CNG channels, making it difficult to detect gating intermediates 1 . In contrast, cAMP is only a partial agonist for SthK, a prokaryotic CNG channel [24][25][26][27] . In agreement with this, cryo-EM structures of WT SthK yielded only closed states even in the presence of saturating cAMP 28 .…”

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confidence: 99%

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Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

et al. 2022

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Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.

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Section: Discussionsupporting

confidence: 89%

Section: Sthk R120a Reveals Multiple Open Statesmentioning

confidence: 99%

Section: Vsd Positioning Allows For Different Gate Expansionsmentioning

confidence: 99%

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confidence: 99%

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Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

et al. 2022

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“…SthK is very well characterized functionally ,, and structurally , and thus represents an ideal model to evaluate the applicability of stBLMs for functional studies of membrane proteins. Furthermore, the function of SthK is dependent on the lipid composition of the membrane . This makes it an ideal protein to demonstrate the utility of tethered membrane systems to study ion channels whose function depends on more complex lipid mixtures.…”

Section: Introductionmentioning

confidence: 99%

Native Function of the Bacterial Ion Channel SthK in a Sparsely Tethered Lipid Bilayer Membrane Architecture

et al. 2023

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The plasma membrane protects the interiors of cells from their surroundings and also plays a critical role in communication, sensing, and nutrient import. As a result, the cell membrane and its constituents are among the most important drug targets. Studying the cell membrane and the processes it facilitates is therefore crucial, but it is a highly complex environment that is difficult to access experimentally. Various model membrane systems have been developed to provide an environment in which membrane proteins can be studied in isolation. Among them, tethered bilayer lipid membranes (tBLMs) are a promising model system providing a solvent-free membrane environment which can be prepared by self-assembly, is resistant to mechanical disturbances and has a high electrical resistance. tBLMs are therefore uniquely suitable to study ion channels and charge transport processes. However, ion channels are often large, complex, multimeric structures and their function requires a particular lipid environment. In this paper, we show that SthK, a bacterial cyclic nucleotide gated (CNG) ion channel that is strongly dependent on the surrounding lipid composition, functions normally when embedded into a sparsely tethered lipid bilayer. As SthK has been very well characterized in terms of structure and function, it is well-suited to demonstrate the utility of tethered membrane systems. A model membrane system suitable for studying CNG ion channels would be useful, as this type of ion channel performs a wide range of physiological functions in bacteria, plants, and mammals and is therefore of fundamental scientific interest as well as being highly relevant to medicine.

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Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology

et al. 2023

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The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (− 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies. Graphical abstract

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Anionic lipids unlock the gates of select ion channels in the pacemaker family

Cited by 25 publications

References 74 publications

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

Native Function of the Bacterial Ion Channel SthK in a Sparsely Tethered Lipid Bilayer Membrane Architecture

Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology

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