Using Biochemical Light-switches to Illuminate Ion Channel Activity


There’s no doubting that optogenetics is an important recent development within the field of neuroscience. Channelrhodopsins, non-mammalian proteins that conduct ions in response to specific wavelengths of light, have now been inserted into various neuronal pathways to demonstrate the use of light to control modalities as diverse as vision, hearing, pain and motor control. In November 2017, The Scientist reported on progress in human clinical trials using channelrhodopsins in combination with viral vectors to restore a degree of function in damaged sensory neurons in response to light1. In a study conducted by Allergan, patients suffering from retinitis pigmentosa were injected with virus carrying the genetic signal to express channelrhodopsins specifically in retinal ganglion cells, bypassing the damaged light-detecting cells of the retina, enabling a rudimentary sense of light-detection in patients that were previously totally blind. Although primarily a safety study, it has shown promising progress in the field which may soon see developments towards treating hearing loss and chronic pain using a similar approach.

However, alongside the use of non-mammalian light-activated proteins to control neuronal activity, an alternative light-based approach has been developing which has direct and immediate usefulness as a tool in the field of drug discovery. The use of light to reversibly deliver ligand to a native protein receptor or ion channel, or ‘optopharmacology’, is the subject of an interesting recent mini-review by Bregestovski, Maleeva and Gorostiza2. For drug discovery, the use of these chemical photo-switches enables the rapid, and most importantly, reversible activation of ion channel function in response to light. Ligand-gated channels are the subject of this particular review, but much work with voltage-gated channels and G-protein-coupled receptors has now also been published. This approach provides a huge step forward from the previous use of caged-compounds in flash photolysis – often used, for example, to study synaptic transmission, but would leave the preparation awash with ligand that was slow to clear by re-uptake/diffusion, meaning difficult and slow ‘one-hit’ experimentation.

At the Ion Channel Modulation Symposium in Cambridge last year (2016), Dirk Trauner spoke about the development of these tools and demonstrated their use in research conducted both within his group and in conjunction with collaborators around the world, showing examples (see figures 1 & 2 below) of the use of photochromic ligands in both their soluble (PCL) and tethered (PTL) forms.

These two forms are defined by the nature of their interaction with target proteins – PCLs are designed to mimic the ligand of a specific receptor but are freely diffusible and may not exhibit sub-type specificity within a tissue. PTLs, as their named suggests, become covalently tethered to their target, usually via naturally-occurring or genetically modified cysteine residues, conferring a much higher degree of selectivity. On exposure to specific wavelengths of light, the molecules photoisomerize between cis-and trans- states, enabling ligand-receptor interaction that triggers either activation or deactivation of the target protein.

Of the two types, PCLs are the simplest to use. Figure 1 shows the impressive degree of temporal control of gained over the function of the capsaicin receptor TRPV1 in the presence of 1uM of a ‘photolipid’ PCL (here, an azobenzene combining the vanilloid head-group of capsaicin with photoswitch-containing fatty-acid chain (AZCA derivatives)) in response to simply varying light stimulation between 350 and 450 nm.

Figure 1: reproduced from Frank et al (2015)3

Sarah 1

Having proved the principle in TRPV1-expressing HEK cells, optical switching in the presence of this PCL was applied to isolated mouse DRG neurones, and c-fibre nociceptive neurones in saphenous nerve preparations, both of which contain native TRPV1 receptors. All showed rapidly reversible non-selective cation conductance in response to the shorter wavelengths of light, translating into nerve depolarisation and action potential firing in native neurones – responses which were absent in TRPV1-/- knockout mice.

Trauner also presented work using PTLs, and show-cased developments made in collaboration with US colleagues to extend the chemical tether to reinforce its chemical stability and reduce the chance of off-target attachment. Their new ‘PORTL’ (photoswitchable orthogonal remotely tethered ligand), shown in figure 2a, was used to demonstrate dual optical control of mGluR2 and GluK2 expressed in the same cell, differentially responding to the specified wavelengths of light (figure 2b).

Figure 2: reproduced from Broichhagen et al (2015)4                 Sarah 2

This elegant chemistry is a powerful tool for studying ion channel-mediated physiology. Its use, for example, to selectively activate or silence particular neurones, or sub-populations of heteromeric channels containing a common tagged subunit, with a degree of spacial and temporal control unachievable with perfusion, enables more qualitative assessment of the interaction between  new possible therapeutic compounds and their target proteins. And like channelrhodopsins, the use of these photochromic ligands as therapies in their own right is also possible and currently being investigated.

Blog by: Sarah Lilley

References:

1 The Scientist Nov 16 2017 article by Shawna Williams

https://www.the-scientist.com/?articles.view/articleNo/50980/title/Optogenetic-Therapies-Move-Closer-to-Clinical-Use/

2Bregestovski, P., Maleeva, G., and Gorostiza, P. (2017) Light-induced regulation of ligand-gated channel activity. British J Pharm, doi: 10.1111/bph.14022.

3Frank, JA., Moroni, M., Moshourab, R., Sumser, M., Lewin, GR., and Trauner, D. (2015) Photoswitchable fatty acids enable optical control of TRPV1. Nature Comms 6, doi:10.1038/ncomms8118

 4Broichhagen, J., Damijonaitis, A., Levitz, J., Sokol, KR., Leippe, P., Konrad, D., Isacoff, EY., and Trauner, D. (2015) Orthogonal optical control of a G protein-coupled receptor with a SNAP-tethered photochromic ligand. ACS Cent Sci 1 383-393, doi: 10.1021/acscentsci.5b00260

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