Learning & memory: Detailed pharmacology of Mg block . Molecular & formal descriptions

1. BMB 170c Prepares You to Contribute to Three Neuroscience Problems that Involve Ion Channels Presenter: Henry Lester 26 May 2009 Learning & memory: Detailed pharmacology of Mg block . Molecular & formal descriptions Nicotine addiction: Cation-π interactions at the nicotine receptor binding site; Selective Chaperoning of nicotine receptors Epilepsy: Engineering Ion Channels

2. Superfamilies of Neurotransmitter-gated Ion Channel Receptors Cys-loop Receptors Nicotinic ACh 5HT-3 GABAA and GABAC Glycine Ionotropic Glutamate Receptors AMPA-type Kainate-type NMDA-type ATP (P2X) Receptors

3. The NMDA receptor is blocked by Mg2+ in a voltage-dependent manner glutamate Mg2+ outside Functioning channel inside -30 mV or more positive Mg2+-blocked channel -60 mV or more negative

4. Na+, Ca2+ -30 mV The NMDA receptor conducts only when 1. The membrane potential is more positive than -30 mV 2. Glutamate is present (intracellular concentrations of glutamate and Mg2+ are nearly irrelevant) Action potential plus glutamate functioning channel outside inside A molecular coincidence detector leading to Na+ and Ca2+ influx, with many intracellular effects Including long-term potention (LTP)

5. Divalent Cations What is the selective advantage that cells maintain Ca2+ at such low levels? Cells made a commitment, more than a billion yr ago, to use high-energy phosphate bonds for energy storage. Therefore cells contain a high internal phosphate concentration. But Ca phosphate is insoluble near neutral pH. Therefore cells cannot have appreciable concentration of Ca2+; they typically maintain Ca2+ at < 10 –8 M. What is the selective advantage that cells don’t use Mg2+ fluxes? The answer derives from considering the atomic-scale structure of a K+ -selective channel (next slide), which received the 2003 Nobel Chemistry Prize:

6. H2O K+ ion carbonyl KcsA structure K+ ions lose their waters of hydration and are co-ordinated by backbone carbonyl groups when they travel through a channel.

7. As ions pass through ligand-gated channels, Hydroxyl side chains partially substitute for waters of hydration Postulated example: Nicotinic receptor ?

8. Time required to exchange waters of hydration The “surface / volume” principle: We know of several Mg2 transporters, but Mg2+ channels apparently exist only in mitochondria & bacteria. Moomaw & Maguire, Physiologist, 2008

9. all molecules begin here at t= 0 units: s-1 Molecular lifetimes State 1 State 2 k21 open closed Concentration of acetylcholine at A synapse (because of acetylcholinesterase, turnover time ~ 100 μs) high 0 Number of open channels ms

10. . . . . the foot-in-the-door scheme current time

11. Model or scheme all molecules begin here at t= 0 State 1 State 2 k21 normal function open closed k23 = k+[Drug] k21 drug blocked simple block open closed k23 = k+[Drug] k21 drug blocked foot-in-the-door open closed k32 Not allowed

12. + n =1 time constant = 1/k21 0 time constant = 1/(k21+ k23) etc

13. Localizing the V-dependent binding / blocking site for Mg2+ in the NMDA channel McMenimen KA, ACS Chem Biol., 2006

14. BMB 170c Prepares You to Contribute to Three Neuroscience Problems that Involve Ion Channels Presenter: Henry Lester 26 May 2009 Learning & memory: Detailed pharmacology of Mg block . Molecular & formal descriptions Nicotine addiction: Cation-π interactions at the nicotine receptor binding site; Selective Chaperoning of nicotine receptors Epilepsy: Engineering Ion Channels

15. Nearly Complete Cys-loop Receptor (February, 2005) ~ 2200 amino acids in 5 chains (“subunits”), MW ~ 2.5 x 106 Binding region Membrane region Colored by secondary structure Colored by subunit (chain) Cytosolic region

16. a4 b2 Ligand-binding domain . . . Until 9 Nov 2005 T S I 18' I L L L L L L F F 13' V V M1 T T L L M2 10' S A 9' L L L M3 L L V V S 6' S I I C C M4 L L T T 2' Intracellular loop The 9’Leucine and 13’Valine residues are conserved among most / all Cys-loop receptor subunits and reside at or near the gate 13’Val 9’Leu Miyazawa, Fujiyoshi, Unwin, Nature 2003

17. Nicotine and ACh act on many of the same receptors, but . . . • 1. Nicotine is highly membrane-permeant. ACh is not. • Ratio unknown, probably > 1000. • 2. ACh is usually hydrolyzed by acetylcholinesterase (turnover rate ~104 /s.) In mouse, nicotine is eliminated with a half time of ~ 10 min. • Ratio: ~105 • EC50 at muscle receptors: nicotine, ~400 μM; ACh, ~ 45 μM. • Ratio, ~10. Justified to square this because nH = 2. Functional ratio, ~100. • For nicotine, EC50(muscle) / EC50(α4β2) = 400 • What causes this difference?

18. The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004) aY198 C2 aW149 B aY93 A aY190 C1 non-aW55 D (Muscle Nicotinic numbering)

19. WT WT, without cation-π interaction Nicotine makes a stronger cation-π interaction with Trp B at α4β2 receptors than at muscle receptors; this partially explains α4β2 receptors’ high binding affinity for nicotine.

20. Nicotine makes a stronger H-bond to a backbone carbonyl at α4β2 than at muscle receptors:With amide to ester substitution,EC50 increases 20-fold vs 1.5-fold Weaker hydrogen bond C2 B A C1 Deleted hydrogen bond D

21. Nicotine EC50 values: Muscle nAChR single component ~ 400 μM α4β2 two components ~ 1 μM, ~200 μM Underlying the 400-fold higher nicotine sensitivity of neuronal vs muscle receptors: Factor of ~16 for the cation-π interaction; Factor of ~ 12 for H-bond; 16 x 12 = 192. We still can’t explain a factor of 400/192 ~ 2. Xiu, Puskar, Shanata, Lester, Dougherty. Nature 2009

22. Changes with chronic nicotine 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).

23. Upregulation is a part of SePhaChARNS Nicotine is a “Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry” Behavior Circuits Synapses Neurons Nicotine Addiction Intracell. Binding Parkinson’s Disease Nic vs ACh ADNFLE Proteins RNA Genes

24. 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 Free Energy Reaction Coordinate #2. Binding eventually favors high-affinity states

25. Thermodynamics of SePhaChARNS, #3. Reversible stabilization amplified by covalent bonds? Covalently stabilized AR*HS ? + nicotine RHS RLS Degradation Nicotine Increased High-Sensitivity Receptors hr 0 20 40 60

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

27. 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) EYFP XFP = mCerulean ECFP mEGFP mVenus mCherry mEYFP Förster resonance energy transfer (FRET): a test for subunit proximity β2 λ→ Neuro2a

28. 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

29. A key SePhaChARNS experiment: changes in subunit stoichiometry caused by chronic nicotine Behavior Circuits Synapses Neurons Nicotine Addiction Intracell. Binding Parkinson’s Disease Nic vs ACh ADNFLE Proteins RNA Genes Neuro2a

30. Differential subcellular localization and dynamics of α4GFP* receptors plasma memb. mCherry overlay α4GFPβ2 overlay α4GFPβ4 (1:1) α4GFPβ2 (1:1) 3 RXR/β subunit α4GFPβ4 (1:1) zero RXR/β subunit

31. BMB 170c Prepares You to Contribute to Three Neuroscience Problems that Involve Ion Channels Presenter: Henry Lester 26 May 2009 Learning & memory: Detailed pharmacology of Mg block Molecular & formal descriptions Nicotine addiction: Cation-π interactions at the nicotine receptor binding site; Selective Chaperoning of nicotine receptors Epilepsy: Engineering Ion Channels

32. Neuronal Engineering with Cys-loop receptors Goal: develop a general technique to selectively and reversibly silence or activate specific sets of neurons in vivo. • Rationale: Investigate functional roles of defined neurons in ways not feasible with present techniques. • Therapy for diseases of excessive neuronal activity, e g epilepsy • Ideal approach would: • Have on- and off- kinetics on a time scale of minutes • Have simple activation (ie, via drug injected or in animal’s diet) • Avoid nonspecific effects in animal • Maintain target neurons healthy in an “off-state” for a few days without morphological/other changes • Silence “diffuse” molecularly defined sets of neurons, not just spatially defined groups

33. The “channelohm” is 2% of the human genome, and many other organisms expand the repertoire Voltage (actually, ΔE ~107 V/m) External transmitter Internal transmitter Light Temperature Force/ stretch/ movement Blockers Binding region Switches Resistor Battery = Membrane region 1/r = 0.1 – 100 pS Nernst potential for Na+, K+, Cl-, Ca2+, H+ Colored by subunit (chain) Cytosolic region (incomplete) Invertebrate glutamate-gated Cl- channel . At this resolution, resembles nicotinic acetylcholine receptor

34. The drugs “avermectins” • IVM: Lactone originally isolated from Streptomyces avermitilis • AVMs are used as antiparasitics in animals and humans (“River blindness” / Heartgard™) • IVM is probably an allosteric activator of GluCl channels • Also modulates GABA, 5HT3, P2X, and nicotinic channels, at much higher doses (IVM) The channel resembles the nicotinic receptor & requires two subunits

35. First tests: HEK cells

36. IVM-induced silencing in GluCl-expressing cultured rat hippocampal neurons 500 nm IVM 50 nm IVM 5 nm IVM

37. Fluorescent Labels in the M3-M4 loop, function is retained A a, YFP; b, CFP ab a ab (FRET shows that the subunits co-assemble)

38. Cation-psidechain Aligns with GluClb Y182 We wish to eliminate possible glutamate sensitivity in GluCl Colored by subunit (chain)

39. b Y182F eliminates glutamate responses but retains IVM responses 1 mm Glu 1 mM IVM

40. Excessive variability among culture dishes

41. A B C D Optimized constructs optGluCla,b = “AVMR-Cl” • Binding site: • subunit unmutated; b Tyr182Phe (cation-π site) • suppresses endogenous glutamate sensitivity • M3-M4 intracellular loop: a YFP; b CFP • allows visualization • Coding region: codons adapted for mammalian expression • ~ 10-fold greater expression

42. AAV-2 constructs injected into mouse striatum; slice experiments Single neurons: correlation between IVM-induced conductance & AP silencing

43. Plans to extend the AVMR system Transfer AVM sensitivity to mammalian glycine receptor  no immune response Tighter AVM binding  increased AVM sensitivity M2 mutations  increased AVM sensitivity • Na+-permeable • selective neuronal activation • Ca2+-permeable • manipulate signal transduction Increased single-channel current  increased AVM sensitivity

44. Im Vm Generating the first AVMR-Na GluCl  WT +  WT Muscle nAChR ND96 0.5 (ND96 + Mannitol) (10 nM IVM) Still too small GluCl  P304/A305E +  WT Still too large (200 nM IVM)