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A major pathway for the movement of small ions across membranes is through channels specific to those ions.
- Na+, K+, Ca++, Cl- are important examples
- As the scientist whose lab determined the potassium channel structure writes (ref.):
"The evolution of the lipid cell membrane solved one problem and created another. It enabled compartmentalization of life's essential ingredients, but it made it almost impossible for charged atoms, the ions, to move into and out of cells."
"The electric field around an ion causes it to be repelled away from the oil-like membrane of cells. The repulsion, known as the dielectric barrier, is so great that nature had to fashion specific mechanisms to get ions across the membrane."
"For rapid, selective transmembrane ion flow—the kind underlying electrical signaling in the nervous system and many other cellular processes—membrane-spanning proteins called ion channels were nature's solution."
Movement of these small ions is related to maintenance of, and transient changes in, the transmembrane potential (voltage).
- Neurons and muscle cells are specialized to take advantage of these channels
- Most (all?) channels have an open and closed configutation
There are a few types of channel and many subtypes of each
- Basic types include voltage-gated, ligand-gated and second-messanger controlled
- Subtypes vary in structural detail and the resultant properties. For example, different voltage-gated potassium channel subtypes have almost a hundredfold diffference in conductance (permeability)
The crystal structure of a potassium channel, the bacterial KcsA, is solved in detail and it serves as the prototype for understanding all potassium channels.
A central issue related to ion channels is how specificity is achieved.
- For example K+ (1.33 Å) is larger than Na+ (0.95 Å), yet Na+ does not normally pass through K+ channels.
Structure-function correlations of ion channels reveal the basis of their selectivity and of the way that other physiological demands can modify channel dynamics, such as conductance rate.
The KcsA potassium channel is a transmembrane channel that forms a pore through the cell membrane that selectively allows the passage of potassium.
- It is present in prokaryotes and eukaryotes in forms that are evolutionarily related. It can be gated by numerous factors, such as transmembrane voltage in neuronal axons.
- It's crystal structure was first solved by Doyle, et. al (1998) in Streptomyces lividens . Their structure (1bl8.pdb) is shown here. It is composed of four identical subunits, each containing 158 residues.
Each of the four identical subunits is comprised of two helices, with the tetramer lying in the membrane like an inverted teepee when viewed from the extracellular side.
The quaternary structure of the potassium channel is stabilized by formation of salt bridges between neighboring subunits. These inter-subunit salt bridges are very important for the integrity of the quaternary structure and account for about half of the total subunit-subunit association energy.
This rendering (from Berneche & Roux, 2001) shows the channel embedded in a phospholipid membrane bathed by 150mM KCl salt (K green, Cl magenta).
Two layers of aromatic amino acids extend into the lipid bilayer at each end of the molecule, near the membrane water interfaces, helping to anchor the tetramer in the membrane.
More on Potassium Selectivity
(Ref. and Ref. for this section)
Binding sites for K+ ions in the KcsA K+ channel. (a)...subunit closest to the viewer removed. Potassium ions (green spheres) bind at four locations in the selectivity filter (yellow) and in the water-filled cavity at the membrane centre (bottom ion). b, Close-up view of the selectivity filter in ball-and-stick representation, with ...key amino acids forming the selectivity filter shown
The narrow 12 Angstrom-long channel (yellow loops) is where selectivity for K+ but not Na+ occurs. The "signature" five amino acids that line the channel "are arranged with their carbonyl oxygen atoms pointed inward towards the ion conduction pathway. This arrangement creates four potential ion-binding sites into which a K+ ion can bind in an essentially dehydrated state, surrounded by eight oxygen atoms from the protein."
- Na+ does not fit as well, so its binding is not energetically favorable.
- In each of the four sites, a K+ ion can potentially be held at the centre of a box, with one plane of four oxygen atoms above and one plane below the ion."
- "Potassium ions diffuse through the channel at rates approaching 108 ions s-1 under physiological conditions. To catalyse such high diffusion rates, the selectivity filter must allow a K+ ion to dehydrate, enter and cross the selectivity filter within about 10 ns."
As shown in the next figure, "the K+ selectivity filter usually contains two resident K+ ions separated by a water molecule. The ion pair moves back and forth in a concerted manner between the 1,3 and 2,4 configurations [lower pathway] until a third ion enters on one side, causing the displacement of an ion on the opposite side [upper pathway]. The structure of the selectivity filter is designed by selection to have a maximum rate of conduction through minimization of the energy difference between the 1,3 and 2,4 ion configurations."
How is the water of hydration stripped off as potassium moves through the channel?
There is a 10 angstrom cavity in the middle of the molecule, just below the filter, shown by the star in the next partial-structure diagram. This keeps the K+ ion in a 'friendly' watery environment, even though it is actually in the 'middle' of the lipid membrane.
Eight water molecules surround the K+ in the central cavity, as shown by this x-ray crystallographic density diagram, where red is water and green K+.
- These water molecules that form an ordered hydration structure are themselves surrounded by many other unordered water molecules that fill the rest of the cavity.
Hydration at the outer face of the channel is also achieved by structures bonding with water molecules that fit into the mouth of the channel, where carbonyl oxygen atoms point into the extracellular space.
KcsA crystalizes in a closed pore configuration. Another potassium channel, MthK was crystallized by the same group, in the open configuration. The two channels are similar enough to allow comparison of their open and closed states.
The cellular side of the closed KcsA channel narrows until the helices of the four subunits are as close togetner as possible. Two of the subunits are shown in this picture.
Comparison of KcsA (red) and MthK (black), seen here end on at the intracellular face, shows the difference between the open and closed configurations.
"In MthK the inner helices are bent and splayed open...the bend in the MthK inner helices occurs at a [glycine] hinge point—a gating hinge—that is located deep within the membrane, just below the selectivity filter."
- "Glycine is unique in its ability to adopt awide range of
main-chain dihedral angles and confers flexibility at specific points in protein structures." - "To convert the KcsA (closed pore) structure into the MthK (opened pore) structure, it appears as though one would simply have to exert a lateral (radial-outward) force on the C-terminal (intracellular) extent of the inner helices; such a force would place a torque on the gating hinge."
- "The open pore is very wide, about 12 A ° at its narrowest point, so that the central cavity becomes essentially continuous with the intracellular solution."
This simulation movie shows the suggested change in shape from closed to open to closed. (double-click movie to play).
This figure shows the postulated open (green) versus closed (red) structure of another channel, Kir6.2, based on the KcsA structure. (ref.)
Ligand Gating the Channel
The MthK potassium channel is gated by the second messanger ion Ca++, which binds to a large cytoplasmic portion of the molecule at the intracellular end of the channel. (ref)
There are two cytoplasmic RCK domains for each of the four subunits, yielding a gating ring of eight identical RCK domains. The gating ring is held together by fixed and flexible contacts. The latter allow the movement associated with the binding of calcium.
"The flexible hinge, which allows movement, occurs at the base of the cleft near to where Ca2+ binds in the MthK channel. One can imagine Ca2+ influencing the structure by binding in the cleft," as proposed in this model and movie.
Based on the KcsA and MthK structures and partial structures of other channels, a model is proposed in which a cytoplasmic N-terminal loop invades the channel to inactivate it. The exact way the loop responds to voltage changes has yet to be worked out.
Scorpion Toxin
Scorpion toxin (agotoxin2) paralyzes victims of a bite by binding to potassium channels and blocking potassium ion flow and thus action potentials that activate the muscles needed to breathe. This figure (from Garcia et al., 2001) shows how the toxin ( red chain ) actually binds to the extracellular side of the channel at the selectivity-filter region. (The channel modeled is from a mouse mutant named Shaker .)
References
Berneche, S., Roux, B. Energetics of ion conduction through the K+ channel. Nature Nov. 1, 2001. 414:73-77
Doyle, DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. Apr 3, 1998; 280(5360):69-77. PMID: 9525859
Garcia, M.L., Gao Y.-D., McManus, O.B., Kaczorowski, G.J. Potassium channels: from scorpion venoms to high-resoluttion structure. Toxicon, June 2001, 39(6):739-748.
Guidoni L, Torre V, Carloni P. Potassium and sodium binding to the outer mouth of the K+ channel. Biochemistry. 1999 Jul 6; 38(27):8599-604. PMID: 10393534
Jiang, Y, Lee, A, Chen, J., Cadene, M., Chait, B.T. & MacKinnon, R. The open pore conformation of potassium channels. Nature. 2002 417:523-26.
Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B.T. & MacKinnon, R. Crystal structure and mechanism of a calcium-gated potassium channel. Nature. 2002. 417:515-522.
G. Loussouarn, T. Rose, and C.G. Nichols. Structural Basis of Inward Rectifying Potassium Channel Gating.Trends in Cardiovascular Medicine. 2002; 12:253–258.
MacKinnon, R. Potassium Channel's Secret. Nature. 1999. 5(10):107-8.
MacKinnon, R., Cohen, S.L., Kuo, A., Lee, A., Chait, B.T. Structural conservation in prokaryotic and eukaryotic potassium channels. Science. April 3, 1998. 280:106-109.
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. PMID: 11689935
Perozo, E, Cortes, D.M., Cuello, L.G. Structural rearrangements underlying K+-channel Activation Gating. Science. July 2, 1999. 285:73-78.
Roux B, MacKinnon R. The cavity and pore helices in the KcsA K+ channel: electrostatic stabilization of monovalent cations. Science. 1999 Jul 2; 285(5424):100-2. PMID: 10390357
Valiyaveetil FI, Zhou Y, MacKinnon R. Lipids in the structure, folding, and function of the KcsA K+ channel. Biochemistry. 2002 Sep 3;41(35):10771-7. PMID: 12196015
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. PubMed ID: 11689936
Zhou, M., Morais-Cabral, J.H., Mann, S. & MacKinnon, R. Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature. 2001. 411:657-661.