Selective Pharmacological Chaperoning of Acetylcholine Receptor Number and Stoichiometry.

1. Selective Pharmacological Chaperoning of Acetylcholine Receptor Number and Stoichiometry. SePhaChARNS, The Movies Focus on a42* (But remember that some α42* receptors contain 5, 6, or β3 subunits) Behavior Circuits Synapses Neurons Nicotine Addiction Intracell. October 2009 Henry Lester Binding Parkinson’s Disease Inadvertent therapeutic effects of chronic nicotine Nic vs ACh ADNFLE Proteins RNA Genes

2. Hypothesis: SePhaChARNS underlies the cellular and subcellular specificity of nicotine-induced upregulation (Symposium Tuesday afternoon) CA EC MH DG IPN Medial Perforant Path Striatum SNc Thalamus, superior colliculus SNr * = upregulation shown with electrophysiology Nashmi et al J Neurosci 2007; Xiao et al, J. Neurosci 2009

3. Possible Mechanism for changes with chronic nicotine: “Upregulation” Chronic exposure to nicotine causes upregulation of nicotinic receptor binding (1983: Marks & Collins; Schwartz and Kellar); Upregulation 1) Involves no change in receptor mRNA level; 2) Depends on subunit composition (Lindstrom, Kellar, Perry). Shown in experiments on clonal cell lines transfected with nAChR subunits: Nicotine seems to act as a “pharmacological chaperone” (Lukas, Lindstrom) or “maturational enhancer” (Sallette, Changeux, & Corringer; Heinemann) or “Novel slow stabilizer” (Green). Upregulation is “cell autonomous” and “receptor autonomous” (Henry).

4. Upregulation is a part of SePhaChARNS Nicotine is a “Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry” • Related phenomena: • 1. Chronic nicotine • ADNFLE mutations • 3. β2 vs β4 subunit • Trafficking motifs • Lynx proteins • Single molecules Neuro2a

5. Bound states with increasing affinity unbound + + + C Highest affinity bound state AC Free Energy A2C A2O A2D Reaction Coordinate Thermodynamics of SePhaChARNS Increasingly stable assembled states Free subunits #1. Nicotine binds to subunit interfaces, favoring assembled receptors (“matchmaking”) Free Energy Reaction Coordinate #2. Binding eventually favors high-affinity states

6. Thermodynamics of SePhaChARNS #3. Acid trapping may keep intracellular nAChRs desensitized Nicotine accumulates in cells 1 mM Nicotine+ (pKa = 7.8) pH 7.4 pH 7.0 2.5 mM Nicotine+ . . . and then in intracellular organelles. P. Paroutis, N. Touret, S Grinstein (2004) Physiology 19: 207-215 nicotine+/nicotine: 10 30 100 300

7. Thermodynamics of SePhaChARNS, #4. Reversible stabilization amplified by covalent bonds? Covalently stabilized AR*HS ? + nicotine RHS RLS Degradation Nicotine Increased High-Sensitivity Receptors hr 0 20 40 60

8. Overview of membrane protein traffic Secretory pathway Pharmacological chaperoning: upregulation starts here (Oversimplification) Early LTP / Opioids: regulation starts here (Oversimplification)

9. M3 - M4 α4 N C N C loop M3 - M4 loop Ligand binding M1 M2 M3 M4 M4 HA tag XFP c - myc tag XFP b a 2 - XFP 4 - XFP FRET pairs (m = monomeric) mEYFP XFP = mCerulean ECFP mEGFP mVenus mCherry Fluorescent AChRs for localization and Förster resonance energy transfer (FRET) β2 Raad Nashmi Rahul Srinivasan λ Fraser Moss Cagdas Son Neuro2a

10. 50% α-CFP, 50% α-YFP 1/4 1/4 1/2 E No FRET b/a =1.62; 1.62-6 = 0.055 1/8 1/8 1/8 100% α3β2 E1 E2 E3 E4 100% α2β3 No FRET 1/8 1/4 1/4 % receptors with α3 Theory of FRET in pentameric receptors with αnβ(5-n)subunits Cagdas Son

11. Whole-cell donor photobleach experiments suggest 24 hr nicotine (1 μM) shifts nAChR stoichiometry to (α4)2(β2)3 α4 plus β2CFP +β2YFP 1:1 α4CFP +α4YFP 1:1 plus β2 Neuro2a Cagdas Son

12. 4 hour nicotine exposure: increased (a4)2(b2)3 assembly in Golgi WHOLE CELL + 1 mM NICOTINE 4 h R2 = 0.998 y0 = 0 xc1 = 8.5 ± 0.18 w1 = 2.4 ± 0.1 A1 = 130438 ± 36122 xc2 = 10.1 ± 0.26 w2 = 2.24 ± 0.14 A2 = 64907 ± 26106 WHOLE CELL No treatment R2 = 0.999 y0 = 0 xc1 = 8.7 ± 0.06 w1 = 2.22 ± 0.12 A1 = 88465 ± 34150 xc2 = 10 ± 0.36 w2 = 2.92 ± 0.19 A2 = 109476 ± 34316 GOLGI No treatment R2 = 0.999 y0 = 0 xc1 = 8.28 ± 0.07 w1 = 1.9 ± 0.05 A1 = 6756 ± 620 xc2 = 9.72 ± 0.07 w2 = 1.8 ± 0.04 A2 = 5298 ± 621 GOLGI + 1 mM NICOTINE 4 h R2 = 0.998 y0 = 0 xc1 = 8.37 ± 0.02 w1 = 2.33 ± 0.03 A1 = 11498 ± 239 xc2 = 10.21 ± 0.04 w2 = 1.51 ± 0.06 A2 = 1986 ± 233 Fraser Moss, Rahul Srinivasan

13. Most membrane proteins exit the ER in a COPII-dependent manner GTPase: Sar1 COP II: Sec23/24 heterodimer scission ER lumen ER membrane Cytosolic compartment Mancias & Goldberg, Traffic 2005

14. ER exit sites and nAChRs Sec24D-eGFP (250 ng) 4-mCherry + 2 (500 ng each) Merge ERES selection Outlines of selected ERES • Transfect N2a cells with nAChRs + ERES marker • Incubate with drug (48 h) • ERES number (Confocal) • ERES intensity (Confocal) • ERES dynamics (TIRFM) Methodology Rigo Pantoja Rahul Srinivasan

15. 48 h drug exposure increases number of ER exit sites (ERES) p < 0.01 P < 0.01 p < 0.05 n = 18 NS n = 30 n = 19 n = 25 Rigo Pantoja Rahul Srinivasan

16. Total internal reflection fluorescence microscopy (TIRFM) highlights events at and near the plasma membrane

17. Plas Mem-mCherry α4GFP-β2 Overlay A. Control TIRFM shows that nicotine encourages nAChRs to exit the ER B. 0.1 µM nic 48 h Rigo Pantoja Rahul Srinivasan Neuro2a

18. The 2 nAChR subunit mediates ER retention of receptors 4eGFP2 4eGFP4 Rigo Pantoja Rahul Srinivasan

19. The2 M3-M4 loop mediates ER retrieval / retention 4eGFP4 + Sec24D-mCherry 4eGFP42i + Sec24D-mCherry Oocyte physiology supports this difference; see also Frahm et al poster Rigo Pantoja Rahul Srinivasan

20. Lynx changes the distribution of α4β2 nAChRs 4GFP β2 nAChR plus Plasma Membrane-cherry Lynx co-transfected cells no additional transfections Organized smooth ER? (also in HEK cells) Julie Miwa Neuro2a

21. Primary neuronal cultures from wild-type vs. lynx1-KO cortices transfected with 4-cherry2 nAChRs Wild-type lynx1-KO Golgi outposts at branch points? Organized smooth ER? Julie Miwa

22. Other fluorescent nAChR subunits α5GFP α4-cherry NFRET

23. Toward single-molecule resolution of α4β2* nAChRs in mammalian cells: receptor clusters and subunit stoichiometry Immobile diffraction- limited puncta α4GFPβ2 (4:1) Photobleaching profile • Criteria for plasma membrane inserted nAChRs • Immobile • Diffraction limited • Discrete photobleaching steps Rigo Pantoja Rahul Srinivasan

24. SePhaChARNS participates in sequelae of chronic exposure to nicotine 1. Nicotine potently activates some neuronal nAChRs (because it participates in both cation-π and H-bond interactions within the conserved aromatic box). 2. This high affinity allows nicotine to act as a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. 3. These processes lead to α4β2* upregulation, with cellular and subcellular specificity. Behavior Circuits 4. Chaperoning may underlie chronic nicotine’s effects on suppression of ADNFLE seizures. Synapses Neurons 5. β2 vs β4 subunits are processed and trafficked differentially and have distinct trafficking motifs in the M3-M4 loops. Intracell. Binding 6. Lynx proteins may also function as chaperones. Nic vs ACh Proteins 7. These phenomena may soon be resolved at the single molecule level. RNA Genes 8. Can these fluorescence assays serve in drug discovery?

25. Dennis Dougherty Nyssa Puskar Jai Shanata Joanne Xiu “Unnatural Club” Crystal Dilworth Ryan Drenan Julie Miwa Elisha Mackey Fraser Moss (Now at Case Western Univ.) Raad Nashmi (now at Univ. Victoria) Rigo Pantoja (now at Life Technologies) Cagdas Son (now at Univ. Ankara) Chris Richards Rahul Srinivasan Cultures: Sheri McKinney, Doreen Rhee “Alpha / Fluorescence Club” Support: NS-11756, Targacept, Louis & Janet Fletcher

26. High-resolution fluorescence microscopy to study SePhaChARNS LTP / Opioids: regulation starts here TIRFM PM ER Pharmacological chaperoning: upregulation starts here FRET Golgi Nucleus