Structure Function of Channels K Roderick

Structure & Function of + Channels K Roderick Mac. Kinnon et al. 1998 - Nobel prize in Chemistry 2003 30. 01. 2007 Lior Golgher Structure & Function of K+ Channels

Motivation – n + K Channels are Essential for neural communication & computation. Voltage-gated ion channels are life’s transistors. n Efficient Block small Na+ ions while letting larger K+ ions flow through. K+ / Na+ affinity >104 without limiting K+ conduction. n Easy to comprehend (but not to investigate). Mostly explained by electrostatic considerations. Separable. ________________ n Elegant 30. 01. 2007 Structure & Function of K+ Channels 2

Agenda n Brief historical background 7 min. n K+ channels structure 15 min. Ion selectivity, voltage sensitivity, high conductance n How was it discovered 8 min. X-ray crystallography, what took 50 years 30. 01. 2007 Structure & Function of K+ Channels 3

Historical background 1/2 n 1855 Ludwig suggests the existence of membranal channels. n 1855 Fick’s diffusion law n 1888 Nernst’s electrodiffusion equation n 1890 Ostwald: Electrical currents in living tissues might be caused by ions moving across cellular membranes. n 1905 Einstein explains brownian motion “Diffusion is like a flea hopping, electrodiffusion is like a flea hopping in a breeze” -- A. L. Hodgkin 30. 01. 2007 Structure & Function of K+ Channels 4

The membrane as an energy barrier n The membrane presents an energy barrier to ion crossing. n Ion pumps build ion concentration gradients. n 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 e. V. 30. 01. 2007 Structure & Function of K+ Channels 5

Historical background n 2/2 1952 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” n 1987 1 st K+ channel sequenced n 1991 K+ channels are tetramers n 1994 Signature sequence identified and linked with selectivity 30. 01. 2007 Structure & Function of K+ Channels 6

Overall structure – Bacterial Kcs. A channel n ~4. 5 nm long, ~1 nm wide (vs. 45 nm @ Intel 2007) n V shaped tetramer n 158 residues n 3 segments: q q q 30. 01. 2007 1. 5 nm Selectivity filter 1. 0 nm Cavity 1. 8 nm Internal pore Structure & Function of K+ Channels 7

Overall structure – Bacterial Kcs. A channel n ~4. 5 nm long, ~1 nm wide (vs. 45 nm @ Intel 2007) n V shaped tetramer n 158 residues n 3 segments: q q q 30. 01. 2007 1. 5 nm Selectivity filter 1. 0 nm Cavity 1. 8 nm Internal pore Structure & Function of K+ Channels 8

Elementary electrostatic considerations n Negative charges raise local K+ availability at channel entrance. n Hydrophobic residues line pore, allowing water molecules to interact strongly with the K+ ion. 30. 01. 2007 Structure & Function of K+ Channels 9

+ K hydration complex in the cavity n A K+ ion is percisely surrounded by 8 water molecules. n High effective K+ conc. (~2 M) at filter entrance. n The four-fold symmetry of the K+ channel fits the fundamental structure of a hydrated K+ ion. 30. 01. 2007 Structure & Function of K+ Channels 10

Carbonyl groups serve as “surrogate water” n Backbone carbonyl oxygen atoms create four K+ binding sites that mimic the water molecules surrounding a hydrated K+ ion. n The energetic cost of dehydration is thereby compensated solely for K+ ions. 30. 01. 2007 Structure & Function of K+ Channels 11

Beautifully elegant selectivity n The fixed filter structure is fine-tuned to accommodate a K+ ion. n It cannot shrink enough to properly bind the smaller Na+ ions. n Therefore, the energetic cost for dehydration is higher for Na+ ions. 266 pm n 190 pm Hence selectivity achieved. 30. 01. 2007 Structure & Function of K+ Channels 12

Convergent evolution – cattle grids! n Humans found a similar solution to a similar problem… n The problem - passing big feet, blocking small feet. n The solution? 1 D only 30. 01. 2007 Structure & Function of K+ Channels 13

The selectivity filter as a Newton’s cradle n The selectivity filter is occupied by two K+ ions alternating between two configurations. n Carbonyl rings can be thought of as K+ holes. 30. 01. 2007 Structure & Function of K+ Channels 14

Highly conserved selectivity filter & cavity n The selectivity filter & the cavity residues are highly conserved through various species and channel types. 30. 01. 2007 Structure & Function of K+ Channels 15

Voltage-gated ion channel superfamily n More than 140 members. n Conductance varies by 100 fold. n Variable gating: voltage, 2 nd messengers, stimuli (p. H, heat, tension, etc. ) n KL Cav Nav n Bacterial ancestor likely similar to Kcs. A channel. 30. 01. 2007 Structure & Function of K+ Channels 16

Voltage gating n 4 positively charged arginine residues on each voltage sensor (~3. 5 e+). n Depolarization inflicts rotation of sensors towards extracellular end of the membrane. n The voltage sensor is mechanically coupled to the outer helix. n Conserved glycine residue serves as a hinge for inner helix. 30. 01. 2007 Structure & Function of K+ Channels 17

2 conduction enhancement mechanisms n Rings of fixed negative charges increase the local concentration of K+ ions at the intracellular channel entrance – from 150 m. M to 500 m. M. n Increasing the inner pore radius reduces its ionophobic barrier height. n Consequently, some K+ channels conduct better than nonselective gap junctions channels. 30. 01. 2007 Structure & Function of K+ Channels 18

And now for the final part 30. 01. 2007 Structure & Function of K+ Channels 19

Revealing the + K channel structure n Mac. Kinnon’s story n X-ray crystallography n Crystallization 30. 01. 2007 Structure & Function of K+ Channels 20

Roderick Mac. Kinnon n Born 1956 n 1978 B. Sc. in Biochemistry @ Brandeis U. n 1981 M. D. @ Tufts U. School of Medicine n 1985 Internal Medicine @ Beth Israel Hospital, Boston n 1987 back to science: post-doc @ Brandeis n 1989 Assoc. prof. @ Harvard U. n 1996 X-ray crystallography @ Rockefeller U. n 1998 K+ channel structure resolved at 0. 32 nm resolution n 2001 30. 01. 2007 0. 2 nm Structure & Function of K+ Channels 21

Neurotoxins shut 30. 01. 2007 + K channels Structure & Function of K+ Channels 22

X-ray Crystallography is just like light Microscopy, except… n Wavelength ~0. 2 nm instead of ~500 nm n No X-ray lenses No imaging – only a spatial Fourier transform of the object. n Incoherent sources No info on phase. n 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. n The inverse Fourier transform has to be calculated based on measured intensities and predicted phases. 30. 01. 2007 Structure & Function of K+ Channels 23

Crystallization with antigen binding fragments n Transmembrane proteins are difficult to crystallize. ~700 / 40000. n Mice Ig. G RNA RT-PCR cloned with E. Coli cleaved with papain n Kcs. A purified with detergent, cleaved with chymotrypsin & mixed with Fab. n Kcs. A-Fab complex crystallized using the sitting-drop method n Papain Fab used as search model. 30. 01. 2007 Structure & Function of K+ Channels 24

Summary n n n 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. 30. 01. 2007 Structure & Function of K+ Channels 25

Questions? 30. 01. 2007 Structure & Function of K+ Channels 26

Hearing is based on 30. 01. 2007 + K Structure & Function of K+ Channels 27

Gate closing leads to filter closing 30. 01. 2007 Structure & Function of K+ Channels 28

Bibliography 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Zhou Y, Morais-Cabral JH, Kaufman A, Mac. Kinnon R. , 'Chemistry of ion coordination and hydration revealed by a K+ channel- Fab complex at 2. 0 A resolution', Nature. 2001 Nov 1; 414(6859): 43 -8. Hodgkin AL, Huxley AF. , 'A quantitative description of membrane current and its application to conduction and excitation in nerve' , J Physiol. 1952 Aug; 117(4): 500 -44. Morais-Cabral JH, Zhou Y, Mac. Kinnon R. , 'Energetic optimization of ion conduction rate by the K+ selectivity filter' , Nature. 2001 Nov 1; 414(6859): 37 -42. Gouaux E, Mackinnon R. , 'Principles of selective ion transport in channels and pumps. ' , Science. 2005 Dec 2; 310(5753): 1461 -5. Mac. Kinnon R. , 'Potassium channels and the atomic basis of selective ion conduction (Nobel Lecture)' , Angew Chem Int Ed Engl. 2004 Aug 20; 43(33): 4265 -77. Hille B. , 'Ionic channels of excitable membranes' , 2 nd edn. , Sinauer Associates, 1992. 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. 387 -95. Doyle D. A. , Morais Cabral J. , Pfuetzner R. A. , Kuo A. , Gulbis J. M. , Cohen S. L. , Chait B. T. , Mac. Kinnon R. , 'The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity', Science. 1998 Apr 3; 280(5360): 69 -77. Chung SH, Allen TW, Kuyucak S. , 'Modeling diverse range of potassium channels with Brownian dynamics' , Biophys J. 2002 Jul; 83(1): 263 -77 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. 2003 Jul 22; 100(15): 9017 -22 Miller C. , 'An overview of the potassium channel family' , Genome Biol. 2000; 1(4): reviews 0004. 1–reviews 0004. 5. Hebert S. C. , Desir G. , Giebisch G. , Wang W. , 'Molecular diversity and regulation of renal potassium channels ', Physiol Rev. 2005 Jan; 85(1): 319 -71. Valiyaveetil FI, Leonetti M, Muir TW, Mackinnon R. , 'Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation', Science. 2006 Nov 10; 314(5801): 1004 -7 Jiang Y, Lee A, Chen J, Ruta V, Cadene M, Chait BT, Mac. Kinnon R. , 'X-ray structure of a voltage-dependent K+ channel', Nature. 2003 May 1; 423(6935): 33 -41 Sigworth F. J. , 'Life's Transistors', Nature. 2003 May 1; 423(6935): 21 -2. Yu F. H. , Catterall W. A. , 'Overview of the voltage-gated sodium channel family' , Genome Biol. 2003 4(3): 207. The Royal Swedish Academy of Sciences, 'Advanced information on the Nobel Prize in Chemistry', 8 October 2003 Mac. Kinnon R. , 'Potassium channels', FEBS Letters, Nov. 2003 555(1) pp. 62 -65 Mac. Kinnon R. , 'Potassium channels', Talk given at C 250 Brain and Mind Symposium in Columbia University, 13 May 2004 Sussman J. L. , ‘Protein Structure & Function 1 – Lecture #9 - Intro. to Protein Crystallography’ , FGS, Weizmann Institute of Science 2007 Jan 9 Hampton Research, ‘Crystal Growth 101 - Crystal Growth Techniques ’, 2001 PDB, OPM & First. Glance in JMol Wikipedia Flickr & Google Images 30. 01. 2007 Structure & Function of K+ Channels 29