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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 ofK+ Channels Roderick MacKinnon et al. 1998 - Nobel prize in Chemistry 2003 Structure & Function of K+ Channels
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 • 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 • 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 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 • 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 • ~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 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 • 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 • 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” • 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 • 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 – 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 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 • 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 • 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 • 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 • 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 Structure & Function of K+ Channels
Revealing the K+ channel structure • MacKinnon’s story • X-ray crystallography • Crystallization Structure & Function of K+ Channels
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… • 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 • 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 • 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? Structure & Function of K+ Channels
Hearing is based on K+ Channels Structure & Function of K+ Channels
Gate closing leads to filter closing Structure & Function of K+ Channels
Neurotoxins shut K+ channels Structure & Function of K+ Channels
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 Structure & Function of K+ Channels
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