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.
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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.
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.
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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
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- Neuroscience
Synchronous neuronal activity is organized into neuronal oscillations with various frequency and time domains across different brain areas and brain states. For example, hippocampal theta, gamma, and sharp wave oscillations are critical for memory formation and communication between hippocampal subareas and the cortex. In this study, we investigated the neuronal activity of the dentate gyrus (DG) with optical imaging tools during sleep-wake cycles in mice. We found that the activity of major glutamatergic cell populations in the DG is organized into infraslow oscillations (0.01–0.03 Hz) during NREM sleep. Although the DG is considered a sparsely active network during wakefulness, we found that 50% of granule cells and about 25% of mossy cells exhibit increased activity during NREM sleep, compared to that during wakefulness. Further experiments revealed that the infraslow oscillation in the DG was correlated with rhythmic serotonin release during sleep, which oscillates at the same frequency but in an opposite phase. Genetic manipulation of 5-HT receptors revealed that this neuromodulatory regulation is mediated by Htr1a receptors and the knockdown of these receptors leads to memory impairment. Together, our results provide novel mechanistic insights into how the 5-HT system can influence hippocampal activity patterns during sleep.
