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Structure & Function of K+ Channels

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1 Structure & Function of K+ Channels
Roderick MacKinnon et al Nobel prize in Chemistry 2003 Lior Golgher 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. Easy to comprehend (but not to investigate). Mostly explained by electrostatic considerations. Separable. ________________________________ Elegant And that’s only in E 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 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 Carl Ludwig, Adolf Fick, Walther Nernst, Wilhelm Ostwald, Julius Bernstein Nernst Potential - The potential level across the cell membrane that exactly opposes net diffusion of a particular ion through the membrane is called the Nernst potential for that ion. The magnitude of the Nernst potential is determined by the ratio of the concentrations of that specific ion on the two sides of the membrane. The greater this ratio, the greater the tendency for the ion to diffuse in one direction, and therefore the greater the Nernst potential required to prevent the diffusion. 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 Intel 2007) V shaped tetramer 158 residues 3 segments: 1.5 nm Selectivity filter 1.0 nm Cavity 1.8 nm Internal pore Streptomyces lividans (KcsA) | Leakage channel 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 Two trans-membrane helices – inner and outer. Plus pore helix. 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. 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. 266 pm 190 pm Structure & Function of K+ Channels

13 Convergent evolution – cattle grids! 1D only
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. TRP = Transient receptor potential. TRPV (vanilloid) are thermometers, TRPM5 (meltasin) taste, CNG = Cyclic nucleotide-gated ion channel. Such as cAMP, cGMP, cADPR. Found in olfactory and visual sensors. 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 Tufts U. School of Medicine 1985 Internal Beth Israel Hospital, Boston 1987 back to science: Brandeis 1989 Assoc. Harvard U. 1996 X-ray Rockefeller U. 1998 K+ channel structure resolved at 0.32 nm resolution 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 / 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 The usual witchcraft – mice & papayas. pUC18 DNA is a commonly used plasmid cloning vector in E.coli. Fab useful as the search model – helps against the phase problem 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
TRPA channel TRP = Transient receptor potential. TRPV (vanilloid) are thermometers, TRPM5 (meltasin) taste Structure & Function of K+ Channels

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

29 Bibliography 30.01.2007 Structure & Function of K+ Channels
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):43-8. 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):37-42. 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, 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 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):69-77. Chung SH, Allen TW, Kuyucak S., 'Modeling diverse range of potassium channels with Brownian dynamics', Biophys J 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 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):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 May 1;423(6935):33-41 Sigworth F.J., 'Life's Transistors', Nature May 1;423(6935):21-2. Yu F.H., Catterall W.A., 'Overview of the voltage-gated sodium channel family', Genome Biol (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 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 Crystal Growth Techniques’, 2001 PDB, OPM & FirstGlance in JMol Wikipedia Flickr & Google Images 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 1013 protein molecules (10-10 moles) 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 Structure & Function of K+ Channels

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

34 APPLETS Structure & Function of K+ Channels

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