Transcript K + Channels - Weizmann Institute of Science

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.

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
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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
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Historical background
1/2

1855 Ludwig suggests the existence of membranal channels.

1855 Fick’s diffusion law

 K   
 K   
k BT
 
 
VK 
 ln   o   26.7 mV  ln   o 
  K  
  K  
q
1888 Nernst’s electrodiffusion equation
i 
i 



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.
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

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:



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 @ 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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Elementary electrostatic considerations
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Negative charges
raise local K+
availability at channel
entrance.

Hydrophobic residues
line pore, allowing
water molecules to
interact strongly with
the K+ ion.
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+
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.
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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.
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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.

190 pm
266 pm
Hence selectivity achieved.
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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.

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.

Carbonyl rings can be thought of as K+ holes.
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Highly conserved selectivity filter & cavity

The selectivity filter & the
cavity residues are highly
conserved through various
species and channel types.
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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.
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Voltage gating
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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.
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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
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MacKinnon’s story
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X-ray crystallography

Crystallization
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Roderick MacKinnon
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Born 1956
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1978 B.Sc. in Biochemistry @ Brandeis U.

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

2001
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0.2 nm
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Neurotoxins shut
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+
K
channels
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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.
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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.
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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 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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