1. MacKinnon, R. Potassium Channels and the Atomic Basis of Selective Ion Conduction. Nobel Lecture, December 8, 2003.
2. Doyle, D.A. et al. The Structure of the Potassium Channel: Molecular basis of K+ Conduction and Selectivity. Science 280: 69-77 (1998).
3. Zhou et al., Chemistry of Ion Coordination and Hydration Revealed by a K+ Channel-FAB Complex at 2.0 Angstroms Resolution. Nature 414:43-48 (2001).
4. Jiang et al., X-ray Structure of a Voltage-Dependent K+ Channel. Nature 423:33-31 (2003).
www.rcsb.org Rutgers Center for Structural Biology. Database of biological structures.
Concise answers to the Study Guide questions and molecular images resulting from the modeling exercises are available on the Proteopedia website. Simply type in ‘autism’ in the search bar.
1. Action Potentials. What are the key conductance properties of sodium and potassium channels required for the propagation of an action potential? If [K+]in = 20 [K+ ]out and [Na+]in = 0.1 [Na+ ]out and [Cl-]in = [Cl- ]out use the Goldman Equation to calculate the resting membrane potential of an axon (use a = 0.03 and b = 0.10 for the relative proportions of Na+ and Cl--). Explain what happens to the sodium and potassium currents when there is a sudden increase in voltage at the axon hillock and how it relates to the generation of an action potential.
2.The Structure of the KcsA Potassium Channel. The central puzzle posed by the potassium pore structure is how it achieves its impressive 104 selectivity for K+ compared with Na+. That is, how can it allow K+ ions to flow passively down their electrochemical gradients through the pore at a very high rate while Na+ ions are virtually excluded? Produce a model (using SPDBV and the PDP coordinates (PDB CODES: 1K4C & 14KD)) for the structures of the KcsA tetrameric pore for both the high and low potassium states. Explain why the crystals contain antibody recognition domains (FAB fragments) directed against the channel subunits.
When you download the coordinates (PDB files) you will notice that the asymmetric unit contains just one copy of a pore subunit bound to the two-chain FAB fragment. To build a model of the tetramer you will need apply four-fold rotational symmetry operators to these coordinates and delete the FAB chains. (This is similar to the exercise in Study Guide No. 4 in which you were asked to construct the SHANK3-SAM ‘ Zn++ sheets’ that are thought to be part of post-synaptic scaffolds). Here is how to do it:
a. Download the PDB coordinates. In the Control Panel, select Chain C. In the EDIT window, click on SAVE ACTIVE RESIDUES IN CURRENT LAYER. Give this PDB file a new name, such as KvsA_Lo[K+]_SubunitA_1K4C.
b. Start a new PROJECT Load in four copies of this saved subunit coordinate file and use EDIT to rename the four layers (subunit A, subunit B, etc --- see p. 21, section 49, of the SPDBV Tutorial).
c. Download the full PDB text file by clicking on the ‘dog-eared’ file ICON on the control bar (see page 67 of the Tutorial). Scroll down the extensive remarks section to REMARK 350, which should be highlighted in red. This describes the transformations of the asymmetric unit needed to construct the biologically relevant form of the structure (tetramer in this case). Subunit A should be in the Graphics Window. Click to Subunit B (layer 2) and select it (residues should already be ‘red’). Now click on the second (BTM2) transformation. A matrix should appear in a window. Pick OK. You will now have two subunits in the Graphics Window related by a 90 degree rotation. Do the same for subunits C and D. You should now have a tetrameric channel in the Display window.
d. Now, simply SAVE CURRENT PROJECT (all layers) and give the file a name, such KvcA_Hi[K+]_tetramer_1K4C.
Repeat the same exercise for the low potassium structure (1K4D).
Depict the molecular structure of the selectivity filter for the high concentration potassium state. What role do the residues in the conserved ‘signature sequence’ (TVGYG) play in defining the chemical characteristics of the filter. What side-chain interactions stabilize the pore loop? Describe the entrance to the selectivity filter and highlight key elements. What happens to a K+ ion as it approaches the channel and enters the selectivity filter? What happens to a K+ ion as it leaves the selectivity filter? What are the structural roles played by the tryptophan pair W67 and W68? (HINT: check out the interaction with one of the sidechains of the signature sequence for a potassium channel). Depict the major differences in structure between the high and low potassium structures. Are there any structured water molecules in the crystal structures? What is their relationship to the bound cations?
Calculate the electrostatic surface potential for KvcA using the program TOOLS option. Can you paint this on a ‘chalky’ solid surface representation of the structure? What does it mean?
3. The Structure of Voltage-Gated Ion Channels. An essential feature of ion channels, reflecting the voltage-dependent conductance changes predicted by the Hodgkin-Huxley model for the propagation of action potentials along axons, is their ability to open or close in response to local changes in trans-membrane potential. Most gated channels have six transmembrane helices (S1-S6), where S5-S6 form pores very similar to the two-helix structure of KvsA reported by MacKinnon in 1998. The structural basis for gating is thought to reside in the 4-helix bundle S1-S4, with the highly charged S4-helix serving in the role of the ‘gating charge’, an element whose movement in response to changes in voltage regulates the pore diameter.
MacKinnon and his group have determined the structure of a gated channel in complex with an FAB fragment, as well as an ‘isolated voltage sensor’ (S1-S4) structure, also bound to an FAB. Construct a model of the tetrameric voltage-gated potassium channel (KvAP) using the asymmetric unit (FAB:KvAP monomer) as your starting point (i.e. repeat the exercise in question 2 above but with PDB CODE: 1ORQ). In addition, in order to prepare for alignment exercises to follow, extract the structure of the ‘voltage sensor’ from its crystal structure where it is bound to an FAB.
How are the S3 and S4 helices different from what had been predicted on the basis of electrophysiological measurements? Using the alignment procedures used last week to compare the apo and bound forms of the SHANK-PDZ structure (Study Guide No. 4), compare the structure of the ‘isolated voltage sensor’ with the structure of S1-S4 of the entire subunit. What residues are involved in the putative transition between these states (HINT: hinge regions and salt-bridges)? The best way to do this is to align helices S2-S4 using the S2 residues only to do the alignment. Note how crystal packing interactions are invoked by MacKinnon and colleagues to develop a model for the gating action of the voltage sensor. Recapitulate these arguments. In other words, what affect do the interactions with the antibody fragment have on the structure of the pore and its surrounding support helices? Produce a model of this effect (HINT: use KcsA structure as a reference)? Is the pore itself conserved in structure? What is the rms difference in the positions of the backbone atoms? Construct a model of the S1-S4 sensor with the ‘paddle’ in its two positions.
What residues constitute the gating charge? Depict this region of the paddle and compute its electrostatic potential surface.
4. Crystallographic Considerations. Rod MacKinnon won the Nobel Prize for Chemistry in 2003, just five years after he published the first structure of an ion channel Read his Nobel Lecture (paper 1) and describe in 500 words or less how he came to determine the crystal structure. Reading paper 2, describe how he used mass spectrometry to help obtain crystals. What method did he use to solve the phase problem? The first structure was of relatively low resolution, but good enough to trace the chain and establish side-chain positions. How did MacKinnon and his group grow crystals that diffract to higher resolution (paper 3)? How did they obtain crystallographic phases for this structure? Calculate B-factor weighted ribbon structures for the original KcsA structure and compare it to KcsA (Hi [K+]). Is it obvious that the second structure is of higher quality? (HINT: Use the COLOR option of the SPDBV program. Select RIBBON and B-Factor coloring options.).
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