Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands
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
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.
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Further reading
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In the striatum, acetylcholine (ACh) neuron activity is modulated co-incident with dopamine (DA) release in response to unpredicted rewards and reward-predicting cues and both neuromodulators are thought to regulate each other. While this co-regulation has been studied using stimulation studies, the existence of this mutual regulation in vivo during natural behavior is still largely unexplored. One long-standing controversy has been whether striatal DA is responsible for the induction of the cholinergic pause or whether DA D2 receptors (D2Rs) modulate a pause that is induced by other mechanisms. Here, we used genetically encoded sensors in combination with pharmacological and genetic inactivation of D2Rs from cholinergic interneurons (CINs) to simultaneously measure ACh and DA levels after CIN D2R inactivation in mice. We found that CIN D2Rs are not necessary for the initiation of cue-induced decrease in ACh levels. Rather, they prolong the duration of the decrease and inhibit ACh rebound levels. Notably, the change in cue-evoked ACh levels is not associated with altered cue-evoked DA release. Moreover, D2R inactivation strongly decreased the temporal correlation between DA and ACh signals not only at cue presentation but also during the intertrial interval pointing to a general mechanism by which D2Rs coordinate both signals. At the behavioral level D2R antagonism increased the latency to lever press, which was not observed in CIN-selective D2R knock out mice. Press latency correlated with the cue-evoked decrease in ACh levels and artificial inhibition of CINs revealed that longer inhibition shortens the latency to press compared to shorter inhibition. This supports a role of the ACh signal and it’s regulation by D2Rs in the motivation to initiate actions.
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