Selective and regulated trapping of nicotinic receptor weak base ligands and relevance to smoking cessation
- University of Chicago, United States
- National Institute of Dental and Craniofacial Research at the National Institutes of Health, United States
- Northwestern University, Feinberg School of Medicine, United States
- Marine Biological Laboratory, United States
Figures
Figure 1 with 3 supplements
Effect of smoking cessation reagents on α4β2R upregulation.
(A) Nicotine, varenicline and lobeline chemical structures. (B) Varenicline and lobeline reduced nicotine upregulation of α4β2Rs for live cells, but not membranes. 125I-epibatidine binding performed …
Figure 1—figure supplement 1
Dose dependence of varenicline and lobeline effects on upregulation.
(A) Dose dependence of varenicline upregulation. α4β2R-expressing cells were treated with increasing concentration of varenicline for 17 hr. Cells were washed with PBS prior to 125I-epibatidine …
Figure 1—figure supplement 2
Altering intracellular pH of acidic vesicles alters the effects of varenicline and lobeline.
(A) 125I-epibatidine binding on α4β2R-expressing HEK cells exposed to varenicline or lobeline 17 hr at 30 μM and then incubated twice (5 min each) with Bafilomycin A (50 nM), an inhibitor of …
Figure 1—figure supplement 3
Release by NH4Cl treatment of α4β2R ligands trapped inside the acidic compartment.
(A) Schematic of the experimental details. (National Center for Chronic Disease Prevention and Health Promotion (US) Office on Smoking and Health, 2014). HEK cells stably expressing α4β2Rs were …
Figure 2
Effect of smoking cessation reagents on functional upregulation.
(A) In this and other recordings, a 17–20 hr treatment with nicotine (Nic, 10 μM) induced a robust, ~5 fold, increase in peak current amplitudes evoked by 1 mM ACh from α4β2R-expressing HEK cells. …
Figure 3 with 1 supplement
α4β2R weak base ligands exhibit different degrees of intracellular trapping.
(A) DHβE, mecamylamine and epibatidine chemical structures. (B) NH4Cl treatment does not alter DHβE (DH) upregulation. 125I-epibatidine binding performed on live α4β2R-expressing cells without …
Figure 3—figure supplement 1
Dose dependence of epibatidine effects on upregulation.
(A) Dose dependence of epibatidine upregulation. α4β2R-expressing cells were treated with increasing concentration of epibatidine for 17 hr. Cells were washed with PBS prior to 125I-epibatidine …
Figure 4 with 1 supplement
Direct measurements of 125I-epibatidine trapping.
(A) The biphasic dissociation of 125I-epibatidine from α4β2R HEK cells measured at 37°C. Bound 125I-epibatidine is normalized to % bound at time 0. The line through the data represents a …
Figure 4—figure supplement 1
Association rate of 125I-epibatidine.
Live cells were bound with 0.1 nM 125I-epibatidine at room temperature for the indicated times. The line through the points represents a least-squares fit to the normalized 125I-epibatidine uptake …
Figure 5 with 1 supplement
Nicotine exposure increased the number of α4β2R-containing acidic vesicles.
(A) Imaging α4β2R-containing acidic vesicles and the effect of nicotine exposure. α4SEP was transfected into the α4β2R-expressing HEK cells. Image (100 x) of 3 merged slices near the cell surface …
Figure 5—figure supplement 1
Subcellular distribution of pHluorin tagged α4 subunit (α4SEP) in α4β2R-expressing HEK cells and cortical neurons.
(A) HEK cells are displayed 36 hr post transfection. Transfected α4SEP subunits (green) and DsRed ER (red) are imaged in untreated (top panel) or nicotine treated (10 μM for 17 hr; bottom panel) …
Tables
Table 1
pKas and Kis for the studied weak base α4β2R ligands
| Weak base | pKa (Basic) | Ki | Trapping |
|---|---|---|---|
| Varenicline (partial agonist) | 9.2 (Unal et al., 2012) | 0.4 nM (Rollema et al., 2010) | Yes |
| Lobeline (partial agonist) | 8.8 (Drugbank, 2016) | four nM (Damaj et al., 1997) | Yes |
| Epibatidine (agonist) | 9.5 (ChEMBL, 2017) | 0.01–0.05 nM (Whiteaker et al., 1998; Badio et al., 1994) | Yes |
| Nicotine (agonist) | 8.0 (Barlow and Hamilton, 1962) | eight nM (Whiteaker et al., 1998) | No |
| DHβE (competitive antagonist) | 7.3 (ChEMBL, 2017) | 0.3 μM (Whiteaker et al., 1998) | No |
| Mecamylamine (noncompetitive antagonist) | 11.2 (Remington and Beringer, 2006; Nangia et al., 1996) | >1 mM (Whiteaker et al., 1998) | No |
