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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

Structure & Function ofK+ Channels

Roderick MacKinnon et al. 1998 -

Nobel prize in Chemistry 2003

Structure & Function of K+ Channels

motivation k channels are
Motivation– K+ Channels are
  • Essential for neural communication & computation.

Voltage-gated ion channels are life’s transistors.

  • Efficient

K+ / Na+ affinity >104 without limiting conduction.

  • Easyto comprehend (but not to investigate).

Mostly explained by electrostatic considerations. Separable.

________________________________

  • Elegant

Structure & Function of K+ Channels

agenda
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

historical background 1 2
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

the membrane as an energy barrier
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

historical background 2 2
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

overall structure bacterial kcsa channel
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

overall structure bacterial kcsa channel8
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

elementary electrostatic considerations
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

k hydration complex in the cavity
K+ hydration complex in the cavity
  • The cavity in the center of the membrane is precisely configured to contain a K+ ion surrounded by 8 water molecules.
  • The cavity achieves a very high effective K+ concentration (~2M) at the entrance to the selectivity filter.
  • Suggestively, the fundamental structure of a hydrated K+ ion gave rise to the four-fold symmetry of the K+ channel.

Structure & Function of K+ Channels

carbonyl groups serve as surrogate water
Carbonyl groups serve as “surrogate water”
  • Backbone carbonyl oxygen atoms create a queue of 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

beautifully elegant selectivity
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

convergent evolution
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

the selectivity filter as a newton s cradle
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

highly conserved selectivity filter cavity
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

voltage gated ion channel superfamily
Voltage-gated ion channel superfamily
  • More than 140 members.
  • Conductance varies by 100 fold. Variable gating.
  • KL Cav  Nav
  • Bacterial ancestor likely similar to KcsA channel.

Structure & Function of K+ Channels

voltage gating
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

2 conduction enhancement mechanisms
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

and now for the final part
And now for the final part

Structure & Function of K+ Channels

revealing the k channel structure
Revealing the K+ channel structure
  • MacKinnon’s story
  • X-ray crystallography
  • Crystallization

Structure & Function of K+ Channels

roderick mackinnon
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

x ray crystallography is just like light microscopy except
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

crystallization with antigen binding fragments
Crystallization with antigen binding fragments
  • 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

summary
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

questions
Questions?

Structure & Function of K+ Channels

hearing is based on k channels
Hearing is based on K+ Channels

Structure & Function of K+ Channels

gate closing leads to filter closing
Gate closing leads to filter closing

Structure & Function of K+ Channels

neurotoxins shut k channels
Neurotoxins shut K+ channels

Structure & Function of K+ Channels

what was known by 1992 hille
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

fine tuning for k conduction
Fine tuning for K+ conduction

Structure & Function of K+ Channels

bibliography
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
  • Hampton Research, ‘Crystal Growth 101 - Crystal Growth Techniques’, 2001
  • PDB, OPM & FirstGlance in JMol
  • Wikipedia
  • Flickr & Google Images

Structure & Function of K+ Channels