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30.01.2007Lior GolgherStructure & Function of K + Channels Roderick MacKinnon et al. 1998 - Nobel prize in Chemistry 2003.

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Presentation on theme: "30.01.2007Lior GolgherStructure & Function of K + Channels Roderick MacKinnon et al. 1998 - Nobel prize in Chemistry 2003."— Presentation transcript:

1 Lior GolgherStructure & Function of K + Channels Roderick MacKinnon et al Nobel prize in Chemistry 2003

2 Structure & Function of K + Channels 2 Motivation – K + Channels are Essential for neural communication & computation. Voltage-gated ion channels are life’s transistors. Efficient Block small Na + ions while letting larger K + ions flow through. K + / Na + affinity >10 4 without limiting K + conduction. Easy to comprehend (but not to investigate). Mostly explained by electrostatic considerations. Separable. ________________________________ Elegant

3 Structure & Function of K + Channels 3 Agenda Brief historical background 7 min. K + channels structure 15 min. Ion selectivity, voltage sensitivity, high conductance How was it discovered 8 min. X-ray crystallography, what took 50 years

4 Structure & Function of K + Channels 4 Historical background1/ Ludwig suggests the existence of membranal channels Fick’s diffusion law 1888 Nernst’s electrodiffusion equation 1890 Ostwald: Electrical currents in living tissues might be caused by ions moving across cellular membranes Einstein explains brownian motion “Diffusion is like a flea hopping, electrodiffusion is like a flea hopping in a breeze” -- A.L. Hodgkin

5 Structure & Function of K + Channels 5 The membrane as an energy barrier The membrane presents an energy barrier to ion crossing. Ion pumps build ion concentration gradients. These concentration gradients are used as an energy source to pump nutrients into cells, generate electrical signals, etc. Born’s equation (1920) - The free energy of transfer of a mole of ion from one dielectric to another: For K + and Na + ions ΔG ≈ 100 Kcal/mole, or ~4 eV.

6 Structure & Function of K + Channels 6 Historical background2/ Hodgkin & Huxley reveal sigmoid kinetics of K + channel gating g K α m 4 “Details of the mechanism will probably not be settled for the time” st K + channel sequenced 1991 K + channels are tetramers 1994 Signature sequence identified and linked with selectivity

7 Structure & Function of K + Channels 7 Overall structure – Bacterial KcsA channel ~4.5 nm long, ~1 nm wide (vs. 45 Intel 2007) V shaped tetramer 158 residues 3 segments:  1.5 nm Selectivity filter  1.0 nm Cavity  1.8 nm Internal pore

8 Structure & Function of K + Channels 8 Overall structure – Bacterial KcsA channel ~4.5 nm long, ~1 nm wide (vs. 45 Intel 2007) V shaped tetramer 158 residues 3 segments:  1.5 nm Selectivity filter  1.0 nm Cavity  1.8 nm Internal pore

9 Structure & Function of K + Channels 9 Elementary electrostatic considerations Negative charges raise local K + availability at channel entrance. Hydrophobic residues line pore, allowing water molecules to interact strongly with the K + ion.

10 Structure & Function of K + Channels 10 K + hydration complex in the cavity A K + ion is percisely surrounded by 8 water molecules. High effective K + conc. (~2M) at filter entrance. The four-fold symmetry of the K + channel fits the fundamental structure of a hydrated K + ion.

11 Structure & Function of K + Channels 11 Carbonyl groups serve as “ surrogate water ” Backbone carbonyl oxygen atoms create four K + binding sites that mimic the water molecules surrounding a hydrated K + ion. The energetic cost of dehydration is thereby compensated solely for K + ions.

12 Structure & Function of K + Channels 12 Beautifully elegant selectivity The fixed filter structure is fine-tuned to accommodate a K + ion. It cannot shrink enough to properly bind the smaller Na + ions. Therefore, the energetic cost for dehydration is higher for Na + ions. Hence selectivity achieved. 190 pm 266 pm

13 Structure & Function of K + Channels 13 Humans found a similar solution to a similar problem… The problem - passing big feet, blocking small feet. The solution? – cattle grids! 1D only Convergent evolution

14 Structure & Function of K + Channels 14 The selectivity filter as a Newton ’ s cradle The selectivity filter is occupied by two K + ions alternating between two configurations. Carbonyl rings can be thought of as K + holes.

15 Structure & Function of K + Channels 15 Highly conserved selectivity filter & cavity The selectivity filter & the cavity residues are highly conserved through various species and channel types.

16 Structure & Function of K + Channels 16 More than 140 members. Conductance varies by 100 fold. Variable gating: voltage, 2 nd messengers, stimuli (pH, heat, tension, etc.) K L  Ca v  Na v Bacterial ancestor likely similar to KcsA channel. Voltage-gated ion channel superfamily

17 Structure & Function of K + Channels 17 Voltage gating 4 positively charged arginine residues on each voltage sensor (~3.5 e + ). Depolarization inflicts rotation of sensors towards extracellular end of the membrane. The voltage sensor is mechanically coupled to the outer helix. Conserved glycine residue serves as a hinge for inner helix.

18 Structure & Function of K + Channels 18 2 conduction enhancement mechanisms Rings of fixed negative charges increase the local concentration of K + ions at the intracellular channel entrance – from 150 mM to 500 mM. Increasing the inner pore radius reduces its ionophobic barrier height. Consequently, some K + channels conduct better than nonselective gap junctions channels.

19 Structure & Function of K + Channels 19 And now for the final part

20 Structure & Function of K + Channels 20 Revealing the K + channel structure MacKinnon’s story X-ray crystallography Crystallization

21 Structure & Function of K + Channels 21 Born B.Sc. in Brandeis U Tufts U. School of Medicine 1985 Internal Beth Israel Hospital, Boston 1987 back to science: Brandeis 1989 Assoc. Harvard U X-ray Rockefeller U K + channel structure resolved at 0.32 nm resolution nm Roderick MacKinnon

22 Structure & Function of K + Channels 22 Neurotoxins shut K + channels

23 Structure & Function of K + Channels 23 X-ray Crystallography is just like light Microscopy, except … Wavelength ~0.2 nm instead of ~500 nm  No X-ray lenses  No imaging – only a spatial Fourier transform of the object. Incoherent sources  No info on phase. Low Luminosity  Weak signal  A crystal structure required  The measured pattern is the product of the reciprocal lattice with the Fourier transform of the electron density map.  The inverse Fourier transform has to be calculated based on measured intensities and predicted phases.

24 Structure & Function of K + Channels 24 Crystallization with antigen binding fragments Transmembrane proteins are difficult to crystallize. ~700 / difficult Mice IgG RNA  RT-PCR  cloned with E.Coli  cleaved with papain KcsA purified with detergent, cleaved with chymotrypsin & mixed with Fab. KcsA-Fab complex crystallized using the sitting-drop method Fab used as search model. Papain

25 Structure & Function of K + Channels 25 Summary K + channels are highly optimized for the selective conductance of K + ions. Selectivity is realized by compensating the energetic cost for K + ions dehydration. Two K + ions oscillate within the filter as in a Newton’s cradle. Negative charges increase the conductance by raising the local K + conc. Positive charges are used for voltage sensing. Separation of properties (selectivity, conductance and gating) allows different channels to use the same mechanisms throughout the tree of life.

26 Structure & Function of K + Channels 26 Questions?

27 Structure & Function of K + Channels 27 Hearing is based on K + Channels

28 Structure & Function of K + Channels 28 Gate closing leads to filter closing

29 Structure & Function of K + Channels 29 Bibliography 1. Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R., 'Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution', Nature Nov 1;414(6859): Hodgkin AL, Huxley AF., 'A quantitative description of membrane current and its application to conduction and excitation in nerve', J Physiol Aug;117(4): Morais-Cabral JH, Zhou Y, MacKinnon R., 'Energetic optimization of ion conduction rate by the K+ selectivity filter', Nature Nov 1;414(6859): Gouaux E, Mackinnon R., 'Principles of selective ion transport in channels and pumps.', Science Dec 2;310(5753): MacKinnon R., 'Potassium channels and the atomic basis of selective ion conduction (Nobel Lecture)', Angew Chem Int Ed Engl Aug 20;43(33): Hille B., 'Ionic channels of excitable membranes', 2nd edn., Sinauer Associates, Yu F.H., Yarov-Yarovoy V., Gutman G.A., Catterall W.A., 'Overview of molecular relationships in the voltage-gated ion channel superfamily', Pharmacol Rev. 57(4), Dec. 2005, pp Doyle D.A., Morais Cabral J., Pfuetzner R.A., Kuo A., Gulbis J.M., Cohen S.L., Chait B.T., MacKinnon R., 'The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity', Science Apr 3;280(5360): Chung SH, Allen TW, Kuyucak S., 'Modeling diverse range of potassium channels with Brownian dynamics', Biophys J Jul;83(1): Brelidze TI, Niu X, Magleby KL., 'A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification', Proc Natl Acad Sci U S A Jul 22;100(15): Miller C., 'An overview of the potassium channel family', Genome Biol. 2000; 1(4): reviews0004.1–reviews Hebert S.C., Desir G., Giebisch G., Wang W., 'Molecular diversity and regulation of renal potassium channels ', Physiol Rev Jan;85(1): Valiyaveetil FI, Leonetti M, Muir TW, Mackinnon R., 'Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation', Science Nov 10;314(5801): Jiang Y, Lee A, Chen J, Ruta V, Cadene M, Chait BT, MacKinnon R., 'X-ray structure of a voltage-dependent K+ channel', Nature May 1;423(6935): Sigworth F.J., 'Life's Transistors', Nature May 1;423(6935): Yu F.H., Catterall W.A., 'Overview of the voltage-gated sodium channel family', Genome Biol (3): The Royal Swedish Academy of Sciences, 'Advanced information on the Nobel Prize in Chemistry', 8 October MacKinnon R., 'Potassium channels', FEBS Letters, Nov (1) pp MacKinnon R., 'Potassium channels', Talk given at C250 Brain and Mind Symposium in Columbia University, 13 May Sussman J.L., ‘Protein Structure & Function 1 – Lecture #9 - Intro. to Protein Crystallography’, FGS, Weizmann Institute of Science 2007 Jan Hampton Research, ‘Crystal Growth Crystal Growth Techniques’, PDB, OPM & FirstGlance in JMol 23. Wikipedia 24. Flickr & Google Images

30 Structure & Function of K + Channels 30 Crystallization issues 1/2 Key parameters varied:  pH  Temperature  Protein concentration  Protein sequence  Which precipitant & concentration Crystals can appear in various condition & vary greatly how they diffract X-rays Useful crystals, ~0.1mm on a side, with 40,000 x 40,000 x 40,000 = 6.4 x protein molecules ( moles)

31 Structure & Function of K + Channels 31 Crystallization issues 2/2 Step 1: Screening  Start with protein as a solution  Trial and error: different precipitants, pH, etc different conditions  Miniaturize: 1 ml protein/expt by hand, 50 nl by robot  Automate Step 2: Grow large crystals  Optimize quantitative parameters (conc, volumes) Step 3: Check whether your crystal diffracts X-rays back

32 Structure & Function of K + Channels 32 Fine tuning for K + conduction

33 Structure & Function of K + Channels 33 What was known by 1992 (Hille) Selectivity filter up,voltage gating down. (Armstrong, 1975) Dehydration necessary. The “surrogate water” idea. Wrong idea about voltage sensor movement. Some idea about pore residues, but poor understanding of selectivity & conduction mechanisms. (Armstrong & Hille, 1998)

34 Structure & Function of K + Channels 34 APPLETS pm.phar.umich.edu/pdb/1r3j.pdb pm.phar.umich.edu/pdb/1r3j.pdb d=1r3j d=1r3j


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