The efficacy of the food-grade antimicrobial xanthorrhizol against Staphylococcus aureus is associated with McsL channel expression.

Background: The emergence and spread of multidrug-resistant Staphylococcus aureus strains demonstrates the urgent need for new antimicrobials. Xanthorrhizol, a plant-derived sesquiterpenoid compound, has a rapid killing effect on methicillin-susceptible strains and methicillin-resistant strains of S. aureus achieving the complete killing of staphylococcal cells within 2 min using 64 µg/mL xanthorrhizol. However, the mechanism of its action is not yet fully understood. Methods: The S. aureus cells treated with xanthorrhizol were studied using optical diffraction tomography. Activity of xanthorrhizol against the wild-type and mscL null mutant of S. aureus ATCC 29213 strain was evaluated in the time-kill assay. Molecular docking was conducted to predict the binding of xanthorrhizol to the SaMscL protein. Results: Xanthorrhizol treatment of S. aureus cells revealed a decrease in cell volume, dry weight, and refractive index (RI), indicating efflux of the cell cytoplasm, which is consistent with the spontaneous activation of the mechanosensitive MscL channel. S. aureus ATCC 29213ΔmscL was significantly more resistant to xanthorrhizol than was the wild-type strain. Xanthorrhizol had an enhanced inhibitory effect on the growth and viability of exponentially growing S. aureus ATCC 29213ΔmscL cells overexpressing the SaMscL protein and led to a noticeable decrease in their viability in the stationary growth phase. The amino acid residues F5, V14, M23, A79, and V84 were predicted to be the residues of the binding pocket for xanthorrhizol. We also showed that xanthorrhizol increased the efflux of solutes such as K+ and glutamate from S. aureus ATCC 29213ΔmscL cells overexpressing SaMscL. Xanthorrhizol enhanced the antibacterial activity of the antibiotic dihydrostreptomycin, which targets the MscL protein. Conclusion: Our findings indicate that xanthorrhizol targets the SaMscL protein in S. aureus cells and may have important implications for the development of a safe antimicrobial agent.

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

A novel force transduction pathway from a tension sensor to the gate in the mechano-gating of MscL channel.

The bacterial mechanosensitive channel of large conductance MscL is activated exclusively by increased tension in the membrane bilayer. Despite many proposed models for MscL opening, its precise mechano-gating mechanism, particularly how the received force at the tension sensor transmits to the gate remains incomplete. Previous studies have shown that along with amphipathic N-terminus located near the cytoplasmic surface of the membrane, Phe78 residue near the outer surface also acts as a "tension sensor," while Gly22 is a central constituent of the "hydrophobic gate." Present study focused on elucidating the force transmission mechanism from the sensor Phe78 in the outer transmembrane helix (TM2) to the gate in the inner transmembrane helix (TM1) of MscL by applying the patch clamp and molecular dynamics (MD) simulations to the wild type MscL channel and its single mutants at the sensor (F78N), the gate (G22N) and their combination (G22N/F78N) double mutant. F78N MscL resulted in a severe loss-of-function, while G22N MscL caused a gain-of-function channel exhibiting spontaneous openings at the resting membrane tension. We initially speculated that the spontaneous opening in G22N mutant might occur without tension acting on Phe78 residue. To test this hypothesis, we examined the (G22N/F78N) double mutant, which unexpectedly exhibited neither spontaneous activity nor activity by a relatively high membrane tension. To understand the underlying mechanism, we conducted MD simulations and analyzed the force transduction pathway. Results showed that the mutation at the tension sensor (F78N) in TM2 caused decreased interaction of this residue not only with lipids, but also with a group of amino acids (Ile32-Leu36-Ile40) in the neighboring TM1 helix, which resulted in an inefficient force transmission to the gate-constituting amino acids on TM1. This change also induced a slight tilting of TM1 towards the membrane plane and decreased the size of the channel pore at the gate, which seems to be the major mechanism for the inhibition of spontaneous opening of the double mutant channel. More importantly, the newly identified interaction between the TM2 (Phe78) and adjacent TM1 (Ile32-Leu36-Ile40) helices seems to be an essential force transmitting mechanism for the stretch-dependent activation of MscL given that substitution of any one of these four amino acids with Asn resulted in severe loss-of-function MscL as reported in our previous work.

Tight hydrophobic core and flexible helices yield MscL with a high tension gating threshold and a membrane area mechanical strain buffer.

The mechanosensitive (MS) channel of large conductance, MscL, is the high-tension threshold osmolyte release valve that limits turgor pressure in bacterial cells in the event of drastic hypoosmotic shock. Despite MscL from Mycobacterium tuberculosis (TbMscL) being the first structurally characterized MS channel, its protective mechanism of activation at nearly-lytic tensions has not been fully understood. Here, we describe atomistic simulations of expansion and opening of wild-type (WT) TbMscL in comparison with five of its gain-of-function (GOF) mutants. We show that under far-field membrane tension applied to the edge of the periodic simulation cell, WT TbMscL expands into a funnel-like structure with trans-membrane helices bent by nearly 70°, but does not break its 'hydrophobic seal' within extended 20 µs simulations. GOF mutants carrying hydrophilic substitutions in the hydrophobic gate of increasing severity (A20N, V21A, V21N, V21T and V21D) also quickly transition into funnel-shaped conformations but subsequently fully open within 1-8 µs. This shows that solvation of the de-wetted (vapor-locked) constriction is the rate-limiting step in the gating of TbMscL preceded by area-buffering silent expansion. Pre-solvated gates in these GOF mutants reduce this transition barrier according to hydrophilicity and the most severe V21D eliminates it. We predict that the asymmetric shape-change of the periplasmic side of the channel during the silent expansion provides strain-buffering to the outer leaflet thus re-distributing the tension to the inner leaflet, where the gate resides.

Modulation of deep neural circuits with sonogenetics.

Noninvasive control of neuronal activity in the deep brain can be illuminating for probing brain function and treating dysfunctions. Here, we present a sonogenetic approach for controlling distinct mouse behavior with circuit specificity and subsecond temporal resolution. Targeted neurons in subcortical regions were made to express a mutant large conductance mechanosensitive ion channel (MscL-G22S), enabling ultrasound to trigger activity in MscL-expressing neurons in the dorsal striatum and increase locomotion in freely moving mice. Ultrasound stimulation of MscL-expressing neurons in the ventral tegmental area could activate the mesolimbic pathway to trigger dopamine release in the nucleus accumbens and modulate appetitive conditioning. Moreover, sonogenetic stimulation of the subthalamic nuclei of Parkinson's disease model mice improved their motor coordination and mobile time. Neuronal responses to ultrasound pulse trains were rapid, reversible, and repeatable. We also confirmed that the MscL-G22S mutant is more effective to sensitize neurons to ultrasound compared to the wild-type MscL. Altogether, we lay out a sonogenetic approach which can selectively manipulate targeted cells to activate defined neural pathways, affect specific behaviors, and relieve symptoms of neurodegenerative disease.

Approaches for the modulation of mechanosensitive MscL channel pores.

MscL was the first mechanosensitive ion channel identified in bacteria. The channel opens its large pore when the turgor pressure of the cytoplasm increases close to the lytic limit of the cellular membrane. Despite their ubiquity across organisms, their importance in biological processes, and the likelihood that they are one of the oldest mechanisms of sensory activation in cells, the exact molecular mechanism by which these channels sense changes in lateral tension is not fully understood. Modulation of the channel has been key to understanding important aspects of the structure and function of MscL, but a lack of molecular triggers of these channels hindered early developments in the field. Initial attempts to activate mechanosensitive channels and stabilize functionally relevant expanded or open states relied on mutations and associated post-translational modifications that were often cysteine reactive. These sulfhydryl reagents positioned at key residues have allowed the engineering of MscL channels for biotechnological purposes. Other studies have modulated MscL by altering membrane properties, such as lipid composition and physical properties. More recently, a variety of structurally distinct agonists have been shown bind to MscL directly, close to a transmembrane pocket that has been shown to have an important role in channel mechanical gating. These agonists have the potential to be developed further into antimicrobial therapies that target MscL, by considering the structural landscape and properties of these pockets.

Mechanosensitive channel MscL induces non-apoptotic cell death and its suppression of tumor growth by ultrasound.

Mechanosensitive channel of large conductance (MscL) is the most thoroughly studied mechanosensitive channel in prokaryotes. Owing to its small molecular weight, clear mechanical gating mechanism, and nanopore forming ability upon opening, accumulating studies are implemented in regulating cell function by activating mechanosensitive channel of large conductance in mammalian cells. This study aimed to investigate the potentials of mechanosensitive channel of large conductance as a nanomedicine and a mechano-inducer in non-small cell lung cancer (NSCLC) A549 cells from the view of molecular pathways and acoustics. The stable cytoplasmic vacuolization model about NSCLC A549 cells was established via the targeted expression of modified mechanosensitive channel of large conductance channels in different subcellular organelles. Subsequent morphological changes in cellular component and expression levels of cell death markers are analyzed by confocal imaging and western blots. The permeability of mitochondrial inner membrane (MIM) exhibited a vital role in cytoplasmic vacuolization formation. Furthermore, mechanosensitive channel of large conductance channel can be activated by low intensity focused ultrasound (LIFU) in A549 cells, and the suppression of A549 tumors in vivo was achieved by LIFU with sound pressure as low as 0.053 MPa. These findings provide insights into the mechanisms underlying non-apoptotic cell death, and validate the nanochannel-based non-invasive ultrasonic strategy for cancer therapy.

"Force-From-Lipids" Dependence of the MscCG Mechanosensitive Channel Gating on Anionic Membranes.

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments.

Dynamic Interactions Between Brilliant Green and MscL Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

The mechanosensitive ion channel of large conductance (MscL) is a promising template for the development of new antibiotics due to its high conservation and uniqueness to microbes. Brilliant green (BG), a triarylmethane dye, has been identified as a new antibiotic targeted MscL. However, the detailed binding sites to MscL and the dynamic pathway of BG through the MscL channel remain unknown. Here, the dynamic interactions between BG and MscL were investigated using solid-state NMR spectroscopy and molecule dynamics (MD) simulations. Residue site-specific binding sites of BG to the MscL channel were identified by solid-state NMR. In addition, MD simulations revealed that BG conducts through the MscL channel via residues along the inner surface of the pore sequentially, in which the strong hydrophobic interactions between BG and hydrophobic residues F23 and I27 in the hydrophobic gate region of the MscL channel are major restrictions. Particularly, it was demonstrated that BG activates the MscL channel by reducing the hydrophobicity of the F23 in the gate region by water molecules that are bound to BG. Taken together, these simulations and experimental data provide novel insights into the dynamic interactions between BG and MscL, based on which new hydrophobic antibiotics and adjuvants targeting MscL can be developed.

Total synthesis of antiviral drug, nirmatrelvir (PF-07321332).

The emergence and rapid spread of coronavirus disease 2019 (COVID-19), a potentially fatal disease, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has swiftly led to public health crisis worldwide. Hence vaccines and antiviral therapeutics are an important part of the healthcare response to combat the ongoing threat by COVID-19. Here, we report an efficient synthesis of nirmatrelvir (PF-07321332), an orally active SARS-CoV-2 main protease inhibitor.

The force-from-lipid principle and its origin, a 'what is true for E. coli is true for the elephant' refrain.

The force-from-lipid (FFL) principle states that it is the lateral stretch force from the lipid membrane that ultimately opens mechanosensitive (MS) channels, not the external tether nor the internal cytoskeleton. Piezo channels for certain touch or proprioception and the hair-cell channels for hearing or balance apparently obey this principle, which is based on the idea that the lipid bilayer is an amphipathic compartment with a distinct internal force-distribution profile. Physical stretch or insertion of chemical impurities alters this profile, driving channel shape change to conform to the new environment. Thus, FFL governs all dynamic proteins embedded in membrane, including Kv's and TRPs. This article retraces the humble origin of the FFL concept. Paramecium research first created the mind set and the resources to electrically explore other microbial membranes. Patch clamp revealed MS-channel activities from yeast and E. coli spheroplasts. Despite formidable obstacles against interdisciplinary research, the E. coli MS-channel protein, MscL, was purified through fractionation by following its activity, much like enzyme purification. Reconstituted into a simple lipid bilayer, pure MscL retains mechanosensitivity, thus firmly establishing the FFL principle in 1994. The relatively simple MscL and its functional cousin MscS soon became ideal models for detailed analyses. Like the DNA-RNA-protein 'central dogma' or ATP synthesis, FFL is a fundamental principle, which appeared early in evolution, retained in all cellular life forms, and is expected to contribute to future molecular research on sensations, homeostasis, and embryonic development.

Photoactivation of a Mechanosensitive Channel.

Optogenetics in the conventional sense, i.e. the use of engineered proteins that gain their light sensitivity from naturally abundant chromophores, represents an exciting means to trigger and control biological activity by light. As an alternate approach, photopharmacology controls biological activity with the help of synthetic photoswitches. Here, we used an azobenzene-derived lipid analogue to optically activate the transmembrane mechanosensitive channel MscL which responds to changes in the lateral pressure of the lipid bilayer. In this work, MscL has been reconstituted in nanodiscs, which provide a native-like environment to the protein and a physical constraint to membrane expansion. We characterized this photomechanical system by FTIR spectroscopy and assigned the vibrational bands of the light-induced FTIR difference spectra of the trans and cis states of the azobenzene photolipid by DFT calculations. Differences in the amide I range indicated reversible conformational changes in MscL as a direct consequence of light switching. With the mediation of nanodiscs, we inserted the transmembrane protein in a free standing photoswitchable lipid bilayer, where electrophysiological recordings confirmed that the ion channel could be set to one of its sub-conducting states upon light illumination. In conclusion, a novel approach is presented to photoactivate and control cellular processes as complex and intricate as gravitropism and turgor sensing in plants, contractility of the heart, as well as sensing pain, hearing, and touch in animals.

Elucidating the molecular basis of spontaneous activation in an engineered mechanosensitive channel.

Mechanosensitive channel of large conductance (MscL) detects and responds to changes in the pressure profile of cellular membranes and transduces the mechanical energy into electrical and/or chemical signals. MscL can be activated using ultrasonic or chemical activation methods to improve the absorption of medicines and bioactive compounds into cells. However, re-engineering chemical signals such as pH change can trigger channel activation in MscL. This study elucidates the activation mechanism of an engineered MscL at an atomic level through a combination of equilibrium and non-equilibrium (NE) molecular dynamics (MD) simulations. Comparing the wild-type (WT) and engineered MscL activation processes suggests that the two systems are likely associated with different active states and different transition pathways. These findings indicate that (1) periplasmic loops play a key role in the activation process of MscL, (2) the loss of various backbone-backbone hydrogen bonds and salt bridge interactions in the engineered MscL channel causes the spontaneous opening of the channel, and (3) the most significant interactions lost during the activation process are between the transmembrane helices 1 and 2 in engineered MscL channel. The orientation-based biasing approach for producing and optimizing an open MscL model used in this work is a promising way to characterize unknown protein functional states and investigate the activation processes in ion channels and transmembrane proteins in general. This work paves the way for a computational framework for engineering more efficient pH-sensing mechanosensitive channels.

2021 Nobel Prize for mechanosensory transduction.

Written by someone who has worked in the mechanobiology field for close to 40 years, this commentary describes some historical background to the recent award of one-half of the Nobel Prize for Physiology or Medicine to Ardem Patapoutian for his discovery of the family of mechanosensitive Piezo ion channels, which function as mechanoreceptors feeling the environment in senses such as touch, pain, and proprioception.

Pocket delipidation induced by membrane tension or modification leads to a structurally analogous mechanosensitive channel state.

The mechanosensitive ion channel of large conductance MscL gates in response to membrane tension changes. Lipid removal from transmembrane pockets leads to a concerted structural and functional MscL response, but it remains unknown whether there is a correlation between the tension-mediated state and the state derived by pocket delipidation in the absence of tension. Here, we combined pulsed electron paramagnetic resonance spectroscopy and hydrogen-deuterium exchange mass spectrometry, coupled with molecular dynamics simulations under membrane tension, to investigate the structural changes associated with the distinctively derived states. Whether it is tension- or modification-mediated pocket delipidation, we find that MscL samples a similar expanded subconducting state. This is the final step of the delipidation pathway, but only an intermediate stop on the tension-mediated path, with additional tension triggering further channel opening. Our findings hint at synergistic modes of regulation by lipid molecules in membrane tension-activated mechanosensitive channels.

Mechanosensitive Channels Mediate Hypoionic Shock-Induced Aminoglycoside Potentiation against Bacterial Persisters by Enhancing Antibiotic Uptake.

Improving the efficacy of existing antibiotics is a promising strategy for combating antibiotic-resistant/tolerant bacterial pathogens that have become a severe threat to human health. We previously reported that aminoglycoside antibiotics could be dramatically potentiated against stationary-phase Escherichia coli cells under hypoionic shock conditions (i.e., treatment with ion-free solutions), but the underlying molecular mechanism remains unknown. Here, we show that mechanosensitive (MS) channels, a ubiquitous protein family sensing mechanical forces of cell membrane, mediate such hypoionic shock-induced aminoglycoside potentiation. Two-minute treatment under conditions of hypoionic shock (e.g., in pure water) greatly enhances the bactericidal effects of aminoglycosides against both spontaneous and triggered E. coli persisters, numerous strains of Gram-negative pathogens in vitro, and Pseudomonas aeruginosa in mice. Such potentiation is achieved by hypoionic shock-enhanced bacterial uptake of aminoglycosides and is linked to hypoionic shock-induced destabilization of the cytoplasmic membrane in E. coli. Genetic and biochemical analyses reveal that MscS-family channels directly and redundantly mediate aminoglycoside uptake upon hypoionic shock and thus potentiation, with MscL channel showing reduced effect. Molecular docking and site-directed mutagenesis analyses reveal a putative streptomycin-binding pocket in MscS, critical for streptomycin uptake and potentiation. These results suggest that hypoionic shock treatment destabilizes the cytoplasmic membrane and thus changes the membrane tension, which immediately activates MS channels that are able to effectively transport aminoglycosides into the cytoplasm for downstream killing. Our findings reveal the biological effects of hypoionic shock on bacteria and can help to develop novel adjuvants for aminoglycoside potentiation to combat bacterial pathogens via activating MS channels.

Control of gene expression in engineered mammalian cells with a programmable shear-stress inducer.

In humans, cellular mechanoperception serves as the basis of touch sensation and proprioception, contributes to the proper programming of cell fate during embryonic development, and plays a pivotal role in the development of mechanosensitive tissues. Molecular mechanoreceptors can respond to their environment by mediating transient adjustments of ion homeostasis, which subsequently trigger calcium-dependent alteration of gene expression via specific signaling pathways such as the nuclear factor of the activated T-cells pathway. Although, mechanoreceptors are potential drug targets for various diseases, current techniques to study mechanically gated processes are often based on custom-tailored microfluidic systems, which require special setups or have limited throughput. Here, we present a platform to characterize shear-stress-triggered, calcium-mediated gene expression, which employs a programmable, 96-well-format, shear-stress induction device to examine the effects of imposing various mechanical loads on mammalian adherent cell lines. The presented method is suitable for high-throughput experiments and provides a large tunable parameter space to optimize conditions for different cell types. Our findings indicate that the device is an effective tool to explore conditions in terms of frequency, intensity, intervals as well as extracellular matrix composition alongside the evaluation of different combinations of mechanosensitive proteins for mechanically activated gene expression. We believe our results can serve as a platform for further investigations into shear stress-controlled gene expression in basic research and drug screening.

A native cell membrane nanoparticles system allows for high-quality functional proteoliposome reconstitution.

Proteoliposomes mimic the cell membrane environment allowing for structural and functional membrane protein analyses as well as antigen presenting and drug delivery devices. To make proteoliposomes, purified functional membrane proteins are required. Detergents have traditionally been used for the first step in this process However, they can irreversibly denature or render membrane proteins unstable, and the necessary removal of detergents after reconstitution can decrease proteoliposome yields. The recently developed native cell membrane nanoparticles (NCMN) system has provided a variety of detergent-free alternatives for membrane protein preparation for structural biology research. Here we attempt to employ the MCMN system for the functional reconstitution of channels into proteoliposomes. NCMN polymers NCMNP1-1 and NCMNP7-1, members of a NCMN polymer library that have been successful in extraction and affinity purification of a number of intrinsic membrane proteins, were selected for the purification and subsequent reconstitution of three bacterial channels: KcsA and the mechanosensitive channels of large and small conductance (MscL and MscS). We found that channels in NCMN particles, which appeared to be remarkably stable when stored at 4 °C, can be reconstituted into bilayers by simply incubating with lipids. We show that the resulting proteoliposomes can be patched for electrophysiological studies or used for the generation of liposome-based nanodevices. In sum, the findings demonstrate that the NCMN system is a simple and robust membrane protein extraction and reconstitution approach for making high-quality functional proteoliposomes that could significantly impact membrane protein research and the development of nanodevices.