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.
Reviewing Editor
- Gary Yellen, Harvard Medical School, United States
Version 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)
- Version of Record updated: December 15, 2022 (version 4)
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
Postsynaptic mitochondria are critical for the development, plasticity, and maintenance of synaptic inputs. However, their relationship to synaptic structure and functional activity is unknown. We examined a correlative dataset from ferret visual cortex with in vivo two-photon calcium imaging of dendritic spines during visual stimulation and electron microscopy reconstructions of spine ultrastructure, investigating mitochondrial abundance near functionally and structurally characterized spines. Surprisingly, we found no correlation to structural measures of synaptic strength. Instead, we found that mitochondria are positioned near spines with orientation preferences that are dissimilar to the somatic preference. Additionally, we found that mitochondria are positioned near groups of spines with heterogeneous orientation preferences. For a subset of spines with a mitochondrion in the head or neck, synapses were larger and exhibited greater selectivity to visual stimuli than those without a mitochondrion. Our data suggest mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs innervating the basal dendrites of cortical neurons.
