Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands
- California Institute of Technology, United States
- Howard Hughes Medical Institute, United States
- University of Bristol, United Kingdom
- Howard Hughes Medical Institute, California Institute of Technology, United States
Abstract
Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ('iDrugSnFRs') for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.
Data availability
Plasmids containing our sensors have been deposited in Addgene (as named in our manuscript) with genetic maps. They are currently viewable and are available on request.The Protein Data Bank has published the crystallographics and structural data (accession codes 7S7T, 7S7U, 7S7V). Supplemntary Table 1 gives relevant details.
-
Crystal structure of iNicSnFR3a Fluorescent Nicotine SensorProtein Data Bank, 7S7V.
Article and author information
Author details
Funding
Tobacco-Related Disease Research Program (Postdoctoral Training Fellowship (27FT-0022))
- Aaron L Nichols
Leiden University International Studies Fund ((LISF L18020-1-45))
- Laura Luebbert
National Institute on Drug Abuse (Exploratory/Developmental Grants (R21) (DA049140))
- Anand K Muthusamy
National Institute of General Medical Sciences (Predoctoral Training in Biology and Chemistry (T32) (GM7616))
- Anand K Muthusamy
Tobacco-Related Disease Research Program (High Impact Pilot Award (27IP-0057))
- Henry A. Lester
Tobacco-Related Disease Research Program (High Impact Research Project Award (T29IR0455))
- Dennis A Dougherty
National Institute of General Medical Sciences (Research Project (GM-123582)
- Henry A. Lester
National Institute on Drug Abuse (Exploratory/Developmental Grants (R21,DA043829))
- Henry A. Lester
Howard Hughes Medical Institute
- Jonathan S Marvin
Howard Hughes Medical Institute
- Loren L. Looger
Howard Hughes Medical Institute
- Douglas C Rees
UK Engineering and Physical Sciences Research Council (No. EP/N024117/1)
- Timothy Gallagher
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: General of primary mouse hippocampal culture was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to the approved institutional animal care and use committee (IACUC) protocols (IA19-1386) of California Institute of Technology. Dissections were performed after euthanasia of the pregnant mouse and every effort was made to minimize suffering.
Reviewing Editor
- Gary Yellen, Harvard Medical School, United States
Publication history
- Preprint posted: October 4, 2021 (view preprint)
- Received: October 12, 2021
- Accepted: January 3, 2022
- Accepted Manuscript published: January 4, 2022 (version 1)
- Version of Record published: February 7, 2022 (version 2)
- Version of Record updated: March 4, 2022 (version 3)
Copyright
© 2022, Nichols et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,311
- Page views
-
- 180
- Downloads
-
- 4
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands
eLife 11:e74648.
https://doi.org/10.7554/eLife.74648
Further reading
-
- Neuroscience
Neurodegenerative diseases are characterized by selective vulnerability of distinct cell populations; however, the cause for this specificity remains elusive. Here we show that entorhinal cortex layer 2 (EC2) neurons are unusually vulnerable to prolonged neuronal inactivity compared with neighboring regions of the temporal lobe, and that reelin+ stellate cells connecting EC with the hippocampus are preferentially susceptible within the EC2 population. We demonstrate that neuronal death after silencing can be elicited through multiple independent means of activity inhibition, and that preventing synaptic release, either alone or in combination with electrical shunting, is sufficient to elicit silencing-induced degeneration. Finally, we discovered that degeneration following synaptic silencing is governed by competition between active and inactive cells, which is a circuit refinement process traditionally thought to end early in postnatal life. Our data suggests that the developmental window for wholesale circuit plasticity may extend into adulthood for specific brain regions. We speculate that this sustained potential for remodeling by entorhinal neurons may support lifelong memory but renders them vulnerable to prolonged activity changes in disease.
-
- Neuroscience
Human behaviour requires flexible arbitration between actions we do out of habit and actions that are directed towards a specific goal. Drugs that target opioid and dopamine receptors are notorious for inducing maladaptive habitual drug consumption; yet, how the opioidergic and dopaminergic neurotransmitter systems contribute to the arbitration between habitual and goal-directed behaviour is poorly understood. By combining pharmacological challenges with a well-established decision-making task and a novel computational model, we show that the administration of the dopamine D2/3 receptor antagonist amisulpride led to an increase in goal-directed or ‘model-based’ relative to habitual or ‘model-free’ behaviour, whereas the non-selective opioid receptor antagonist naltrexone had no appreciable effect. The effect of amisulpride on model-based/model-free behaviour did not scale with drug serum levels in the blood. Furthermore, participants with higher amisulpride serum levels showed higher explorative behaviour. These findings highlight the distinct functional contributions of dopamine and opioid receptors to goal-directed and habitual behaviour and support the notion that even small doses of amisulpride promote flexible application of cognitive control.
