Transcript Structure and function of K channels (MacKinnon)

Structure & Function of
+
K Channels
Roderick MacKinnon et al. 1998 Nobel prize in Chemistry 2003
30.01.2007 Lior Golgher
Structure & Function of K+ Channels
Motivation – K+ Channels are

Essential for neural communication & computation.
Voltage-gated ion channels are life’s transistors.
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Efficient
K+ / Na+ affinity >104 without limiting conduction.
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Easy to comprehend (but not to investigate).
Mostly explained by electrostatic considerations.
Separable.
________________________________
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Elegant
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Agenda
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Brief historical background
7 min.
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K+ channels structure
15 min.
Ion selectivity, voltage sensitivity, high conductance

How was it discovered
8 min.
X-ray crystallography, what took 50 years
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Historical background
1/2
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1855 Ludwig suggests the existence of membranal channels.
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1855 Fick’s diffusion law
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 K   
 K   
k BT
 
 
VK 
 ln   o   26.7 mV  ln   o 
  K  
  K  
q
1888 Nernst’s electrodiffusion equation
i 
i 


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1890 Ostwald: Electrical currents in living tissues might be caused
by ions moving across cellular membranes.
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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
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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:
z 2e2 N A  1 1 
G 
    water  80,  lipid  2
8 0 r   2 1 
For K+ and Na+ ions ΔG ≈ 100 Kcal/mole, or ~4 eV.
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Historical background
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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”
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1987 1st K+ channel sequenced
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1991 K+ channels are tetramers
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1994 Signature sequence identified
and linked with selectivity
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Overall structure – Bacterial KcsA channel
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~4.5 nm long, ~1 nm wide
(vs. 45 nm @ Intel 2007)
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V shaped tetramer
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158 residues
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3 segments:
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

1.5 nm Selectivity filter
1.0 nm Cavity
1.8 nm Internal pore
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Overall structure – Bacterial KcsA channel
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~4.5 nm long, ~1 nm wide
(vs. 45 nm @ Intel 2007)
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V shaped tetramer
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158 residues
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3 segments:



1.5 nm Selectivity filter
1.0 nm Cavity
1.8 nm Internal pore
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Elementary electrostatic considerations
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Negative charges
raise local K+
availability at channel
entrance.
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Hydrophobic residues
line pore, allowing
water molecules to
interact strongly with
the K+ ion.
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+
K
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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.
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Carbonyl groups serve as “surrogate
water”
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Backbone carbonyl oxygen atoms
create a queue of K+ binding sites
that mimic the water molecules
surrounding a hydrated K+ ion.
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The energetic cost of dehydration
is thereby compensated solely for
K+ ions.
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Beautifully elegant selectivity
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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 266 pm
for Na+ ions.
Hence selectivity
achieved.
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190 pm
12
Convergent evolution – cattle grids!
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Humans found a similar solution to a similar problem…
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The problem - passing big feet, blocking small feet.
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The solution?
1D only
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The selectivity filter as a Newton’s cradle
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The selectivity filter is occupied by two K+
ions alternating between two configurations.
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Carbonyl rings can be thought of as K+ holes.
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Highly conserved selectivity filter & cavity
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The selectivity filter & the
cavity residues are highly
conserved through various
species and channel types.
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Voltage-gated ion channel superfamily
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More than 140 members.
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Conductance varies by
100 fold. Variable gating.
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KL  Cav  Nav
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Bacterial ancestor likely
similar to KcsA channel.
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Voltage gating
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4 positively charged arginine residues on
each voltage sensor (~3.5 e+).
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Depolarization inflicts rotation of sensors
towards extracellular end of the
membrane.
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The voltage sensor is mechanically
coupled to the outer helix.
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Conserved glycine residue serves as a
hinge for inner helix.
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2 conduction enhancement mechanisms
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Rings of fixed negative charges increase the
local concentration of K+ ions at the intracellular
channel entrance – from 150 mM to 500 mM.
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Increasing the inner pore radius reduces its
ionophobic barrier height.
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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
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MacKinnon’s story
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X-ray crystallography
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Crystallization
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Roderick MacKinnon
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Born 1956
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1978 B.Sc. in Biochemistry @ Brandeis U.
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1981 M.D. @ Tufts U. School of Medicine
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1985 Internal Medicine @ Beth Israel Hospital, Boston
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1987 back to science: post-doc @ Brandeis
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1989 Assoc. prof. @ Harvard U.
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1996 X-ray crystallography @ Rockefeller U.
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1998 K+ channel structure resolved at 0.32 nm resolution
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2001
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X-ray Crystallography is just like light
Microscopy, except…
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Wavelength ~0.2 nm instead of ~500 nm
 No X-ray lenses  No imaging – only a
spatial Fourier transform of the object.
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Incoherent sources  No info on phase.
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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.
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Crystallization with antigen binding fragments
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Mice IgG RNA  RT-PCR  cloned
with E.Coli  cleaved with papain
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KcsA purified with detergent, cleaved
with chymotrypsin & mixed with Fab.
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KcsA-Fab complex crystallized using
the sitting-drop method
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Fab used as search model.
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Papain
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Summary
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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.
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Questions?
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Hearing is based on
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+
K
Structure & Function of K+ Channels
Channels
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Gate closing leads to filter closing
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Neurotoxins shut
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K
channels
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What was known by 1992 (Hille)
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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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Fine tuning for
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K
conduction
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Bibliography
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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
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2.
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3.
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.
4.
Gouaux E, Mackinnon R., 'Principles of selective ion transport in channels and pumps.', Science. 2005 Dec 2;310(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 20;43(33):426577.
6.
Hille B., 'Ionic channels of excitable membranes', 2nd edn., Sinauer Associates, 1992.
7.
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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 3;280(5360):69-77.
9.
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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
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11.
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12.
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13.
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
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 1;423(6935):33-41
15.
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16.
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17.
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18.
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19.
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20.
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21.
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22.
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23.
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