Journal: The Journal of General Physiology

Article Title: Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors

doi: 10.1085/jgp.201812201

Figure Lengend Snippet: Strategy of the experiments. (A) Nicotine (pKa, 7.5–8.1) and varenicline (pKa, 9.5–10) are weak bases. They interconvert on a millisecond time scale between protonated and deprotonated forms; these are respectively membrane impermeant and permeant. (B) The tactic of confining a genetically encoded fluorescent nicotinic drug sensor to the PM or the ER. (C) Choline, ACh, and N’MeNic exist only as charged, membrane-impermeant forms near physiological pH.

Article Snippet: Online supplementary information includes text information that amine-containing buffers produce anomalous results and that acidic vesicles are candidates for the “sequestered compartment;” Table S1 shows steps in structure and refinement of iNicSnFR1 crystallized with nicotine; Table S2 shows parameters for nicotine and varenicline Matlab/SimBiology models; Fig. S1 shows sequences of PBPs and constructs described in this paper and/or studied in preliminary experiments; Fig. S2 shows directed evolution of the iNicSnFR family; Fig. S3 shows photoswitching noticeable at high [nicotine] with focused laser illumination; Fig. S4 shows responses to nicotine with iNicSnFR_ER in SH-SY5Y cells and HEK293 cells; Fig. S5 shows human iPSCs, differentiated to dopaminergic neurons, transduced with AAV_iNicSnFR3b_ER; and Fig. S6 shows varenicline at iNicSnFR3a expressed in HeLa cells.

Techniques: Membrane

Journal: The Journal of General Physiology

Article Title: Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors

doi: 10.1085/jgp.201812201

Figure Lengend Snippet: Varenicline activity against purified iNicSnFR3a (A–C) and iNicSnFR3b expressed in hippocampal neurons (D–G). (A) Dose–response relations for varenicline-induced ΔF/F 0 , measured in 3× PBS, pH 7.4. Mean ± SEM; three measurements. (B) Stopped-flow measurements at various [varenicline]. (C) Isothermal titration calorimetry. (D–G) Exemplar varenicline-induced fluorescence increases for cultured hippocampal neurons transduced with AAV2/1. syn1 .iNicSnFR3b_PM (D and E) or with AAV2/1. syn1 .iNicSnFR3b_ER (F and G). (D and F) 30-s varenicline pulses, followed by 40-s wash in HBSS. The average waveform for five cells at each [varenicline] is overlaid for the PM and ER traces in A and C, respectively. The SEM is shown as colored bands around each line. (E and G) Dose–response relations, fitted to a single-component Hill equation, including zero response at zero [varenicline]. Parameters are shown.

Article Snippet: Online supplementary information includes text information that amine-containing buffers produce anomalous results and that acidic vesicles are candidates for the “sequestered compartment;” Table S1 shows steps in structure and refinement of iNicSnFR1 crystallized with nicotine; Table S2 shows parameters for nicotine and varenicline Matlab/SimBiology models; Fig. S1 shows sequences of PBPs and constructs described in this paper and/or studied in preliminary experiments; Fig. S2 shows directed evolution of the iNicSnFR family; Fig. S3 shows photoswitching noticeable at high [nicotine] with focused laser illumination; Fig. S4 shows responses to nicotine with iNicSnFR_ER in SH-SY5Y cells and HEK293 cells; Fig. S5 shows human iPSCs, differentiated to dopaminergic neurons, transduced with AAV_iNicSnFR3b_ER; and Fig. S6 shows varenicline at iNicSnFR3a expressed in HeLa cells.

Techniques: Activity Assay, Purification, Isothermal Titration Calorimetry, Fluorescence, Cell Culture, Transduction

Journal: The Journal of General Physiology

Article Title: Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors

doi: 10.1085/jgp.201812201

Figure Lengend Snippet: Simulated pharmacokinetics and pharmacodynamics during oral varenicline administration. (A) In the pharmacokinetic model for orally administered varenicline, the lungs are replaced by the digestive tract. The parameters derive from studies on humans . The parameters are given in Table S2 and in Supplemental ZIP File. (B1) Varenicline concentrations in two compartments: the plasma/CSF/ER and the sequestered compartment. (B2) Effects on the two processes shown in A. Note that recommended treatment with varenicline almost completely activates nAChR protein chaperoning (inside-out process), but only slightly produces nAChR channel activation (outside-in process).

Article Snippet: Online supplementary information includes text information that amine-containing buffers produce anomalous results and that acidic vesicles are candidates for the “sequestered compartment;” Table S1 shows steps in structure and refinement of iNicSnFR1 crystallized with nicotine; Table S2 shows parameters for nicotine and varenicline Matlab/SimBiology models; Fig. S1 shows sequences of PBPs and constructs described in this paper and/or studied in preliminary experiments; Fig. S2 shows directed evolution of the iNicSnFR family; Fig. S3 shows photoswitching noticeable at high [nicotine] with focused laser illumination; Fig. S4 shows responses to nicotine with iNicSnFR_ER in SH-SY5Y cells and HEK293 cells; Fig. S5 shows human iPSCs, differentiated to dopaminergic neurons, transduced with AAV_iNicSnFR3b_ER; and Fig. S6 shows varenicline at iNicSnFR3a expressed in HeLa cells.

Techniques: Drug discovery, Clinical Proteomics, Activation Assay