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

Shivange, Amol V. and Borden, Philip M. and Muthusamy, Anand K. and Nichols, Aaron L. and Bera, Kallol and Bao, Huan and Bishara, Ishak and Jeon, Janice and Mulcahy, Matthew J. and Cohen, Bruce and O'Riordan, Saidhbhe L. and Kim, Charlene and Dougherty, Dennis A. and Chapman, Edwin R. and Marvin, Jonathan and Looger, Loren and Lester, Henry A. (2019) Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors. Journal of General Physiology . ISSN 0022-1295. (In Press) http://resolver.caltech.edu/CaltechAUTHORS:20190204-103658076

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Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1085/jgp.201812201DOIArticle
http://jgp.rupress.org/content/early/2019/02/01/jgp.201812201PublisherArticle
ORCID:
AuthorORCID
Muthusamy, Anand K.0000-0003-1041-914X
Nichols, Aaron L.0000-0001-9341-0049
Bishara, Ishak0000-0001-7563-1980
Jeon, Janice0000-0002-8483-586X
Dougherty, Dennis A.0000-0003-1464-2461
Chapman, Edwin R.0000-0001-9787-8140
Marvin, Jonathan0000-0003-2294-4515
Lester, Henry A.0000-0002-5470-5255
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.
Funders:
Funding AgencyGrant Number
NIHDA036061
NIHDA037161
NIHDA043829
NIHGM123582
NIH Predoctoral FellowshipGM007616
NIHMH061876
NIHNS097362
NIHNS034407
California Tobacco-Related Disease Research Program23XT-0007
California Institute for Regenerative Medicine (CIRM)EDUC2-08398
Brain and Behavior Research FoundationUNSPECIFIED
National Alliance for Research on Schizophrenia and DepressionUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Della Martin FoundationUNSPECIFIED
Louis and Janet FletcherUNSPECIFIED
Caltech Summer Undergraduate Research Fellowship (SURF)UNSPECIFIED
Record Number:CaltechAUTHORS:20190204-103658076
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20190204-103658076
Official Citation:Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors. Amol V. Shivange, Philip M. Borden, Anand K. Muthusamy, Aaron L. Nichols, Kallol Bera, Huan Bao, Ishak Bishara, Janice Jeon, Matthew J. Mulcahy, Bruce Cohen, Saidhbhe L. O'Riordan, Charlene Kim, Dennis A. Dougherty, Edwin R. Chapman, Jonathan Marvin, Loren Looger, Henry A. Lester. The Journal of General Physiology. Feb 2019, jgp.201812201; DOI: 10.1085/jgp.201812201
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:92625
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:04 Feb 2019 18:58
Last Modified:04 Feb 2019 18:58

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