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Published June 2019 | Supplemental Material + Published
Journal Article Open

Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors


Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell–derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as ∼10 nM. Such concentrations would persist during the 12 h of a typical smoker's day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

Additional Information

© 2019 Shivange et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Submitted: 1 August 2018; Revision received 5 November 2018; Accepted: 9 January 2019. Published February 4, 2019. Henry A. Lester and Amol V. Shivange thank the Janelia Research Campus for devoting hospitality and resources in the Visiting Scientist Program over a period of 3 y. We also thank Crystal N. Dilworth (early suggestion to use ProX-family PBPs), Jacob P. Keller (advice on pH and tubing), David P. Walton (analysis of N'MeNic), Michael R. Post (synthesis of N'MeNic), Peter H. Lee and Scott Sternson (purification of N'MeNic), Adela Nano and Jacqueline Barton (access to the ISS-K2 spectrofluorometer), Daniel Wagenaar (construction of LED light sources), Lauren M. Barnett (advice on photochemistry), Elizabeth K. Unger and Lin Tian (biosensors), Eric L. Snapp (ER imaging), Jennifer Lippincott-Schwartz (ER imaging), Eric R. Schreiter (biosensors), Tanner Lakin (cell culture), Kim Ruoho and Melissa Ramirez (AAV constructs), Baljit S. Khakh (advice), Mark Lobas (advice), Victoria J. Orphan and Fabai Wu (use and instruction on structured illumination microscope), Stephen Grant (assisted in confocal microscopy), Margaret Jefferies (excellent laboratory management at Janelia), and Purnima Deshpande (excellent laboratory management at the California Institute of Technology). This research was supported by grants from US National Institutes of Health (DA036061, DA037161, DA043829, GM123582, GM007616, MH061876, NS097362, and NS034407), the California Tobacco-Related Disease Research Project (23XT-0007), the California Institute for Regenerative Medicine (EDUC2-08398), the Brain and Behavior Research Foundation (National Alliance for Research on Schizophrenia and Depression), the Howard Hughes Medical Institute, the Della Martin Foundation, Louis and Janet Fletcher, and California Institute of Technology SURF donors Paraskeva N. Danailov and Maria Chan. E.R. Chapman is an Investigator of the Howard Hughes Medical Institute. H.A. Lester previously had a consulting agreement with Pfizer, who manufacture varenicline. The authors declare no further competing financial interests. Author contributions: A.V. Shivange, P.M. Borden, A.K. Muthusamy, A.L. Nichols, K. Bera, H. Bao, I. Bishara, M.J. Mulcahy, B. Cohen, E.R. Chapman, J. Marvin, L. Looger, and H.A. Lester designed experiments. A.V. Shivange, P.M. Borden, A.K. Muthusamy, A.L. Nichols, K. Bera, H. Bao, I. Bishara, J. Jeon, M.J. Mulcahy, B. Cohen, S.L. O'Riordan, C. Kim and H.A. Lester performed experiments. J. Jeon performed simulations. A.V. Shivange, P.M. Borden, A.K. Muthusamy, J. Jeon, K. Bera, H. Bao, I. Bishara, M.J. Mulcahy, L. Looger, and H.A. Lester wrote the paper. A.V. Shivange, P.M. Borden, A.K. Muthusamy, A.L. Nichols, K. Bera, H. Bao, I. Bishara, J. Jeon, M.J. Mulcahy, B. Cohen, D.A. Dougherty, E.R. Chapman, L. Looger, and H.A. Lester revised the paper. Merritt C. Maduke served as editor.

Attached Files

Published - 738.full.pdf

Supplemental Material - JGP_201812201_DataS1.zip

Supplemental Material - JGP_201812201_V1.mp4

Supplemental Material - JGP_201812201_V2.mp4

Supplemental Material - JGP_201812201_V3.mp4

Supplemental Material - JGP_201812201_V4.mp4

Supplemental Material - JGP_201812201_V5.mp4

Supplemental Material - JGP_201812201_V6.mp4

Supplemental Material - JGP_201812201_V7.mp4

Supplemental Material - JGP_201812201_V8.mp4

Supplemental Material - JGP_201812201_V9.mp4

Supplemental Material - JGP_201812201_sm.pdf


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August 19, 2023
October 20, 2023