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Structure & Function of K + Channels

Structure & Function of K + Channels. Roderick MacKinnon et al. 1998 - Nobel prize in Chemistry 2003. Motivation – K + Channels are. Essential for neural communication & computation. Voltage-gated ion channels are life’s transistors. Efficient

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Structure & Function of K + Channels

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  1. Structure & Function ofK+ Channels Roderick MacKinnon et al. 1998 - Nobel prize in Chemistry 2003 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 >104 without limiting K+ conduction. • Easyto comprehend (but not to investigate). Mostly explained by electrostatic considerations. Separable. ________________________________ • Elegant Structure & Function of K+ Channels

  3. Agenda • Brief historical background7 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 Structure & Function of K+ Channels

  4. Historical background 1/2 • 1855 Ludwig suggests the existence of membranal channels. • 1855 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. • 1905 Einstein explains brownian motion “Diffusion is like a flea hopping, electrodiffusion is like a flea hopping in a breeze”-- A.L. Hodgkin 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. Structure & Function of K+ Channels

  6. Historical background 2/2 • 1952 Hodgkin & Huxley reveal sigmoid kinetics of K+ channel gating gK α m4 “Details of the mechanism will probably not be settled for the time” • 1987 1st K+ channel sequenced • 1991 K+ channels are tetramers • 1994 Signature sequence identified and linked with selectivity Structure & Function of K+ Channels

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

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

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

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

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

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

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

  21. Roderick MacKinnon • Born 1956 • 1978 B.Sc. in Biochemistry @ Brandeis U. • 1981 M.D. @ Tufts U. School of Medicine • 1985 Internal Medicine @ Beth Israel Hospital, Boston • 1987 back to science: post-doc @ Brandeis • 1989 Assoc. prof. @ Harvard U. • 1996 X-ray crystallography @ Rockefeller U. • 1998 K+ channel structure resolved at 0.32 nm resolution • 2001 0.2 nm Structure & Function of K+ Channels

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

  24. Crystallization with antigen binding fragments • Transmembrane proteins are difficult to crystallize. ~700 / 40000. • 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 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. Structure & Function of K+ Channels

  26. Questions? Structure & Function of K+ Channels

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

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

  29. Bibliography • 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. 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, MacKinnon 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. • MacKinnon 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', 2nd 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., MacKinnon 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): reviews0004.1–reviews0004.5. • 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, MacKinnon 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 • MacKinnon R., 'Potassium channels', FEBS Letters, Nov. 2003  555(1) pp. 62-65 • MacKinnon R., 'Potassium channels', Talk given at C250 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 & FirstGlance in JMol • Wikipedia • Flickr & Google Images Structure & Function of K+ Channels

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