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Structure & Function of K + Channels

Explore the structure and function of potassium channels. Learn about their historical background, ion selectivity, and elegant mechanisms that enable neural communication. Discover the beauty of ion dehydration and channel selectivity.

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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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