30.01.2007Lior Golgher - PowerPoint PPT Presentation

Title: 30.01.2007Lior Golgher


1
Structure Function of K Channels

• Roderick MacKinnon et al. 1998 -
• Nobel prize in Chemistry 2003

2
Motivation K Channels are

• Essential for neural communication computation.
• Voltage-gated ion channels are lifes
  transistors.
• Efficient
• Block small Na ions while letting larger K
  ions flow through.
• K / Na affinity gt104 without limiting K
  conduction.
• Easy to comprehend (but not to investigate).
• Mostly explained by electrostatic
  considerations.
• Separable.
• ________________________________
• Elegant

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
Historical background 1/2

• 1855 Ludwig suggests the existence of membranal
  channels.
• 1855 Ficks diffusion law
• 1888 Nernsts 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

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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.
• Borns equation (1920) - The free energy of
  transfer of a mole of ion from one dielectric to
  another
• For K and Na ions ?G 100 Kcal/mole, or 4 eV.

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Historical background 2/2

• 1952 Hodgkin Huxley reveal sigmoid kinetics of
  K channel gating gK a 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

7
Overall structure Bacterial KcsA channel

• 4.5 nm long, 1 nm wide
• (vs. 45 nm _at_ Intel 2007)
• V shaped tetramer
• 158 residues
• 3 segments
• 1.5 nm Selectivity filter
• 1.0 nm Cavity
• 1.8 nm Internal pore

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Overall structure Bacterial KcsA channel

• 4.5 nm long, 1 nm wide
• (vs. 45 nm _at_ Intel 2007)
• V shaped tetramer
• 158 residues
• 3 segments
• 1.5 nm Selectivity filter
• 1.0 nm Cavity
• 1.8 nm Internal pore

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
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
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
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
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
14
The selectivity filter as a Newtons cradle

• The selectivity filter is occupied by two K ions
  alternating between two configurations.
• Carbonyl rings can be thought of as K holes.

15
Highly conserved selectivity filter cavity

• The selectivity filter the cavity residues are
  highly conserved through various species and
  channel types.

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.

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
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.

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And now for the final part
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Revealing the K channel structure

• MacKinnons story
• X-ray crystallography
• Crystallization

21
Roderick MacKinnon

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

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Neurotoxins shut 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
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
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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 Newtons 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
Questions?
27
Hearing is based on K Channels
28
Gate closing leads to filter closing
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. 2001 Nov 1414(6859)43-8.
2. Hodgkin AL, Huxley AF., 'A quantitative
   description of membrane current and its
   application to conduction and excitation in
   nerve', J Physiol. 1952 Aug117(4)500-44.
3. Morais-Cabral JH, Zhou Y, MacKinnon R.,
   'Energetic optimization of ion conduction rate by
   the K selectivity filter', Nature. 2001 Nov
   1414(6859)37-42.
4. Gouaux E, Mackinnon R., 'Principles of selective
   ion transport in channels and pumps.', Science.
   2005 Dec 2310(5753)1461-5.
5. MacKinnon R., 'Potassium channels and the atomic
   basis of selective ion conduction (Nobel
   Lecture)', Angew Chem Int Ed Engl. 2004 Aug
   2043(33)4265-77.
6. Hille B., 'Ionic channels of excitable
   membranes', 2nd edn., Sinauer Associates, 1992.
7. 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.
8. 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
   3280(5360)69-77.
9. Chung SH, Allen TW, Kuyucak S., 'Modeling diverse
   range of potassium channels with Brownian
   dynamics', Biophys J. 2002 Jul83(1)263-77
10. 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 22100(15)9017-22
11. Miller C., 'An overview of the potassium channel
    family', Genome Biol. 2000 1(4)
    reviews0004.1reviews0004.5.
12. Hebert S.C., Desir G., Giebisch G., Wang W.,
    'Molecular diversity and regulation of renal
    potassium channels ', Physiol Rev. 2005
    Jan85(1)319-71.
13. Valiyaveetil FI, Leonetti M, Muir TW, Mackinnon
    R., 'Ion selectivity in a semisynthetic K
    channel locked in the conductive conformation',
    Science. 2006 Nov 10314(5801)1004-7
14. 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
    1423(6935)33-41
15. Sigworth F.J., 'Life's Transistors', Nature. 2003
    May 1423(6935)21-2.
16. Yu F.H., Catterall W.A., 'Overview of the
    voltage-gated sodium channel family', Genome
    Biol. 2003 4(3) 207.
17. The Royal Swedish Academy of Sciences, 'Advanced
    information on the Nobel Prize in Chemistry', 8
    October 2003
18. MacKinnon R., 'Potassium channels', FEBS Letters,
    Nov. 2003  555(1) pp. 62-65
19. MacKinnon R., 'Potassium channels', Talk given at
    C250 Brain and Mind Symposium in Columbia
    University, 13 May 2004

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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)

31
Crystallization issues 2/2

• Step 1 Screening
• Start with protein as a solution
• Trial and error different precipitants, pH,
  etc.100-1000 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
Fine tuning for K conduction
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)

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APPLETS

• http//molvis.sdsc.edu/fgij/fg.htm?molhttp//opm.
  phar.umich.edu/pdb/1r3j.pdb
• http//opm.phar.umich.edu/webmol.php?pdbid1r3j