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2 Thursday 4/20. Lecture 12. Central dogma, part 2. The G protein pathway Thursday-Friday 4/ Sections meet as usual. Monday 4/24 11 AM. Problem Set 4 due, in the Bi 1 Closet. Monday 4/24. Lecture 13. Recreational drugs. Tuesday 4/25. Lecture 14. Central dogma, part 3. Last lecture to be covered on the midterm. Midterm posted. Thursday 4/27. Lecture 15. The human genome. 4 PM: Bi 1 Review session. 119 Kerckhoff (Bi1 Lecture room) Thursday-Friday 4/ Sections meet as usual. No problem set; midterm reviews. Monday 5/1. No lecture. Work on the midterm. Monday 5/1. Midterm due, 4 PM. The Bi 1 Closet Tuesday 5/2. Lecture 16; Problem Set 5 posted. Bi 1 during the next 2 weeks. See also the Bi1 Web Page

3 Bi 1 Lecture 12 Thursday, April 20, 2006 The Central Dogma of Drugs and the Brain, Part 2: Drugs Act on the G Protein Pathway receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

4 Proof of chemical synaptic transmission, 1921 Vagus nerve runs from the head to the heart Spontaneous heartbeats in both hearts are stopped by stimuli to the “upstream” vagus smoked drum The diffusible substance: acetylcholine acting on muscarinic ACh receptors

5 cytosol vesicles containing serotonin vesicles containing dopamine synaptic cleft G protein-coupled (muscarinic) acetylcholine receptor G protein-coupled dopamine receptor cytosol vesicles containing acetylcholine G protein-coupled serotonin receptor Some postsynaptic membranes contain G protein-coupled receptors rather than ligand-gated channels

6 On a time scale of seconds, the language of the nervous system is still electricity; and the central dogma still describes a set of mechanisms that manipulate impulse frequencies in individual neurons.

7 GTPGDP + P i Effector: enzyme or channel outside Neurotransmitter or hormone binds to receptor  activates G protein The central dogma of drugs and the brain, Part 2. Drugs act on the G protein pathway. How fast? 100 ms to 10 s How far? Probably less 1  m      inside

8 1. All have 7  -helices 2. There are about 1000 G protein-coupled receptors in the genome. (Most are still “orphans”; their ligands are unknown) 3. Individual receptors respond to: (a) ) a low-molecular weight neurotransmitter such as serotonin, dopamine, or acetylcholine (b) a short protein (8-40 amino acids, a “peptide”) such as an endorphin (c) an olfactory stimulus; or (d) light, in the eye (receptor = rhodopsin) G protein-coupled receptors receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

9 Selective advantage of such a complex pathway? The neurotransmitter or hormone does not directly influence the response--from the viewpoint of (a)Chemistry (b)Speed (c)Localization (to some extent) All this amplification and indirect coupling requires energy! receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

10 Note the “propeller” in the  subunit which caps the  subunit, preventing either subunit from interacting with the effector (There is no effector in this structure): Swiss-pdb viewer required receptor tsqi G protein effector intracellular messenger 1/G protein-alpha-beta-gamma.pdb Structure of a heterotrimeric G protein: a molecular switch -1/G protein-GDP.pdb 1/G protein-beta-only.pdb Our first real look at ATP / GTP / GDP:

11 GDP Binding site on receptor Agonist G protein Agonist binds succesfully; GDP is replaced by GTP; G protein leaves receptor; G protein activates effector GTP  GDP G protein awaiting  GDP receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector phosphate released to cytosol

12 G protein  Protein G

13 How ”tight” is the gigaohm seal? 1. Electrically tight R =  l/A R ~ 10 9   = resistivity = 22  -cm; l = length = 1  m; A = area = 10  m x t (thickness); Therefore t ~ 2 x m, or less than 1 Å! Little Alberts 12-22A © Garland 1  m From Lecture 7

14 acetylcholine in the pipette opens channels in the pipette 2. Chemically tight The seal compartmentalizes molecules. Molecules outside the pipette do not mix with molecules inside the pipette acetylcholine outside the pipette opens channels outside the pipette How ”tight” is the gigaohm seal? From Lecture 7

15 How ”tight” is the gigaohm seal? 3a. Mechanically tight With strong suction, the patch breaks, but the seal remains intact. Little Alberts 12-22A © Garland 1  m Strong suction From Lecture 8

16 K + Channels are Gi protein effectors 3b. Mechanically tight Use weak suction. Excised “inside-out” patch allows access to the inside surface of the membrane transmitter no GTP n= 0 (closed) no channel openings +G  Normally: released from Gi; Here: added by experimenter +G  n = 0 (closed) n = 1 (open) receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

17 from Lecture 6: Channels are miniature conductors that add in parallel  Na KK mostly K + mostly Na + E Na (+60 mV) G Na =  Na = G  G K =  K E K (-60 mV) G K  K outside inside E Na (+60 mV) G Na =  Na = G  G K =  K E K (-60 mV) E K (- G K  K receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

18 from Lecture 6: 1 ms 5 mV additional K + channels keep the membrane potential away from threshold, and therefore decrease firing rates  V EGEGEG G GG KKNa Cl K NaCl    resting potential C E G Na + K+K+ Cl - outside inside receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

19 Enzyme Ca 2+ in cytosol Enzymes are Gq, Gs, and Gt protein effectors Ca 2+ in vesicles (not synaptic vesicles) Gq receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

20 Gs Enzymes are Gq, Gs, and Gt protein effectors cyclic AMP (cAMP) ATP Enzyme “cyclase” Mg 2+ receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

21 Enzyme “cyclase” Breakdown enzyme “phosphodiesterase” uninteresting molecule caffeine prolongs the intracellular messenger cAMP Inhibited by caffeine receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

22 cAMP ATP Effector enzyme “cyclase” Breakdown enzyme “phosphodiesterase” uninteresting molecule Inhibited by caffeine intracellular messenger Ca 2+ cAMP cGMP GTP Enzyme “cyclase” Breakdown enzyme “phosphodiesterase” uninteresting molecule cGMP Phosphodiesterase inhibitors prolong the life of intracellular messengers Inhibited by Viagra, Cialis, Levitra receptor tsqi G protein enzymechannel effector

23 Many drugs are enzyme inhibitors

24 Selective advantage of such a complex pathway? The neurotransmitter or hormone does not directly influence the response--from the viewpoint of (a)Chemistry (b)Speed (c)Localization (to some extent) All this amplification and indirect coupling requires energy! Further advantages? Suggestions in class: Discussion receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

25 Diversity of the Central Dogma, Part 2 G proteins all have 3 subunits There are ~ 18  subunit genes in 4 major classes i, q, s, t ~ 5  subunits ~ 3  subunits There are 2 major types of effectors Channels affected by G proteins: ~5 known K channel genes ~4 Ca 2+ channels Enzymes 3 major classes, each with 2 to 10 members and many “accessory proteins” ~ 1000 G protein-coupled receptors All have 7 helices receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector

26 End of Lecture 12 receptor tsqi G protein cAMP Ca 2+ intracellular messenger enzymechannel effector