Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.
Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.Gloeobacter violaceus | allosteric effector | evolution | fusion protein | membrane protein P entameric ligand-gated ion channels (pLGICs) mediate chemo-electric signal transduction in animals, thereby fulfilling key physiological functions, including neurotransmission. They are composed of five identical or homologous subunits and carry one to five agonist binding sites on their extracellular domain (ECD) that govern the opening/closing motion of the ion channel within the transmembrane domain (TMD, composed of four helices labeled M1-M4). In human, ≈40 genes code for pLGIC subunits that are organized into two phylogenetic subclasses: cationic excitatory channels, such as nicotinic acetylcholine (nAChR) and 5HT 3 receptors, and anionic inhibitory channels, such as glycine and GABA A receptors (1). More recently, several new members of the family have been discovered in prokaryotes (2), such as the homologous protein from Gloeobacter violaceus (GLIC), which forms a homopentamer functioning as a proton-gated ion channel (3).Eukaryotic and prokaryotic receptors clearly display homologous structures, characterized by a highly conserved β-sandwichfolded ECD coupled to an all-helix TMD, as described by electron microscopy observation of the Torpedo nAChR (TnAChR) (4), and the X-ray structures of the acetylcholine binding protein (...
Activation of stable boron species with visible-light allows the creation of boryl and/or carbon radicals through single electron- or energy transfer.
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