A ligand-gated synthetic ion channel functioning in a living cell

Development of synthetic molecular machines functioning in biological media is one of the challenges of chemistry. We demonstrate ligand-gated ion transportation of a synthetic molecule responding to agonistic and antagonistic ligands not only in an artificial but also in a live-cell membrane.

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Transmembrane proteins are the central molecular machines1 that perform membrane-mediated biochemical functions such as signal transduction, substance transportation and energy conversion.  They also relate to various diseases and infection, thus are important targets not only for fabrication of nanodevices but also for pharmaceutical research.  Inspired by the proteinic channels, for example, numerous types of synthetic ion channels have been developed and used for applications such as sensing in artificial membrane systems.  Yet, it is still a challenge to functionalize the synthetic ion channels in biological membranes.

Fig. 1: Models of a synthetic ion channel embedded in a lipid bilayer complexed with (left) an agonistic ligand and (right) an antagonistic ligand.

Based on our proven molecular design of stimuli-responsive synthetic transmembrane ion channels,2,3 we attempted to demonstrate ligand-gated ion transportation by a totally synthetic molecule embedded in a plasma membrane of a living cell.  For this purpose, we tackled two major problems.  One is to develop methodology to control the orientation of the synthetic molecule in a bilayer membrane.  By combining unsymmetrical molecular design to put phosphate groups at one side of the molecule and post-loading method as a kinetic processing to insert the molecule into a pre-formed bilayer, we succeeded in unidirectional membrane-insertion of the synthetic ion channel.  The second challenge was to characterize the localization of the synthetic molecule in a live-cell plasma membrane.  The membrane localization was directly visualized by state-of-the-art total internal reflection fluorescence (TIRF) microscopy that allowed for real-time single molecule observation.  Eventually, we successfully demonstrated ligand-gated Ca2+ flow into live L cells through the synthetic ion channel.4  Interestingly, analogous to β-adrenergic receptors, the synthetic channel responded to 2-phenylethylamine and propranolol as agonistic and antagonistic ligands, respectively, that bind to different sites of the synthetic ion channel to activate and inhibit the ion transportation capability.

With the techniques of unidirectional orientation of a synthetic transmembrane molecule and its functionalization in a live-cell membrane, operations of synthetic molecular machines in biological media are expected to regulate activities of organelle, cells and tissues in the future.

References:

  1. Feynman, R. P. There's plenty of room at the bottom. Eng. Sci. 23, 22–36 (1960).
  2. Muraoka, T. et al. Reversible ion transportation switch by a ligand-gated synthetic supramolecular ion channel. J. Am. Chem. Soc. 136, 15584–15595 (2014).
  3. Muraoka, T. et al. Mechano-sensitive synthetic ion channels. J. Am. Chem. Soc. 139, 18016–18023 (2017).
  4. Muraoka, T. et al. A synthetic ion channel with anisotropic ligand response. Nat. Commun. 11, 2924 (2020).

Takahiro Muraoka

Associate Professor, Tokyo University of Agriculture and Technology

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