1. K + Channels Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. Biochemistry of Metabolism

2. Voltage-gated K + channels mediate outward K + currents during nerve action potentials. Important advances in understanding have come from:  physiological studies, including the use of patch clamping  mutational studies of the Drosophila voltage-gated K + channel protein, product of the Shaker gene  crystallographic analysis of the structure of bacterial K + channels.  molecular dynamics modeling of permeation dynamics. 4 identical copies of the K + channel protein, arranged as a ring, form the channel walls.

3. Hydropathy analysis & topology studies predicted the presence of 6 transmembrane  -helices in the voltage-gated K + channel protein. The core of the channel consists of helices 5 & 6 & the intervening H5 segment of each of the 4 copies of the protein.

4. Helices 1-4 function as a voltage-sensing domain, with helix #4 having a special role in voltage sensing. This domain is absent in K + channels that are not voltage-sensitive.

5. The selectivity filter that determines which cation can pass through a channel is located at the narrowest part. Mutation studies showed that the H5 segment is essential for K + selectivity. H5 includes a consensus sequence (Thr-Val-Gly-Tyr-Gly) found in all K + channels, with only minor changes through evolution. Selectivity: K + channels are highly selective for K +, e.g., relative to Na +.

6. In this channel protein:  There are only 2 transmembrane  -helices, corresponding to helices # 5 & 6 in the voltage-gated channel.  An intervening segment includes the K + channel consensus sequence. The first K + channel for which an X-ray crystal structure was solved is the KcsA K + channel from Streptomyces lividans.

7.  The K + channel consensus sequence (in black) forms the selectivity filter at the narrowest part of the channel.  K + ions are bound within the selectivity filter (color pink).  The gate is where the innermost  -helices of the 4 subunits meet at the narrow end of the "teepee" structure. This is the closed channel. Left: side view, normal to membrane. Right: view from the extra- cellular space.

8. Most are backbone oxygens of the consensus sequence. Each bound K + interacts with 8 oxygen atoms, as it sits between 2 of the rings of oxygen atoms. See webpage of R. MacKinnon for additional diagrams.webpage As K + enters the channel, its waters of hydration are replaced by oxygen atoms lining the selectivity filter. 4 K + binding sites within the selectivity filter are defined by 5 rings of oxygen atoms. Each ring includes 4 oxygen atoms, 1 from each subunit.

9. The arrangement of O atoms surrounding each K + within the selectivity filter mimics that of a hydrated K + ion. Thus the energy barrier for entry & exit of K + is low. A K + ion is seen closely interacting with the outermost ring of oxygen atoms.

10. Based on predicted electrostatic repulsion & other evidence, K + is assumed to occupy actually every other binding site, with a water in each intervening site. K + & H 2 O alternately pass single file through the channel. As a K + ion binds at one end of the selectivity filter, a K + ion exits at the other end. Solved X-ray structures such as at right, which average over many copies of the channel, show K + in all 4 binding sites.

11. Na +, being smaller than K +, would be able to interact with oxygen atoms on only one side of the channel. Thus removal of the waters of hydration from Na +, for entry into the channel, would be less favored energetically. Furthermore, K + within the selectivity filter is required to stabilize its ion-conducting conformation. If Na + is substituted for K + in the medium, the selectivity filter undergoes a conformational change to a partly collapsed state.

12. The structure of a Ca ++ -dependent K + channel from Methanobacterium thermoautrophicum, MthK, was the first to be solved in the open state, providing information about the mechanism of channel gating. The selectivity filter (K + channel consensus sequence) is colored black in the diagram above. Channel Gating:

13. Channel opening is associated with a bend at a glycine residue in the innermost helix of each copy of the MthK channel protein. In the mammalian voltage-gated K + channel, a Pro-Val-Pro (PXP) motif in the same helix functions as the gating hinge. In contrast to the teepee shape of the closed KcsA channel, the outward splaying of bent helices provides a large opening at the cytosolic end of the open MthK channel.

14. MthK includes a large extracellular protein assembly involved in regulation of gating by Ca ++. The Ca ++ -binding gating ring does not itself block ion flow. It induces channel conformational changes of gating.

15. Voltage sensing: Mutational analysis showed ( + ) residues in helix #4 to be essential for voltage gating. In helix #4 every 3rd residue is Arg or Lys, & intervening residues are hydrophobic. Decreased transmembrane potential causes helix #4 to change position, resulting in more of its (+) charges being accessible to the aqueous phase outside the cell. A small "gating current" is measurable, as (+) charges effectively move outward.

16. Crystal structures have been determined for:  a bacterial voltage-gated K + channel KvAP  a mammalian equivalent of the Shaker channel designated Kv1.2. The core of both voltage-gated channels (selectivity filter & two transmembrane  -helices of each of four copies of the protein) is similar to that of other K + channels. On the next slide is shown the bacterial KvAP channel, in the open conformation.

17. Some  -helices of the voltage-sensing domain of the differ from the expected trans-membrane orientation. The (+) charged voltage-sensing helix #4, & part of what was designated helix #3, associate in a helix-turn-helix to form a "paddle" shape at the periphery of the channel.

18. Fab fragments of monoclonal antibodies raised to the voltage-sensor domain of the KvAP channel protein were used to promote crystallization of the KvAP channel. The structure of the mammalian equivalent of the Shaker channel has been determined in the absence of FAB fragments but in complex with a regulatory protein. In this channel,  -helices of the voltage-sensing domain are tilted relative to the plane of the membrane but have a more transmembrane orientation. For diagrams see: website (Brookhaven Nat. Lab)website articlearticle (Long et al., Science).

19. Some differences between solved KvAP & Shaker channel structures are postulated to be due to altered conformation of the KvAP channel on being removed from the lipid membrane. A genetically engineered chimaeric channel, in which the voltage sensor paddle of another K + channel is incorporated into the Shaker-type Kv1.2 channel, has been crystallized in the presence of lipids. The structure of this channel in a lipid membrane-like environment is found to be similar to that of the previously solved Kv1.2 structure.

20. According to current models, a voltage change drives movement of each positively charged voltage sensor paddle complex across the membrane. This exerts tension, via a linker segment, on the end of each inner helix of the channel core to promote bending, and thus channel opening. Recent high-resolution structural studies permit predictions of how acidic residues may stabilize positive charges on the paddle as it moves within the membrane. Diagram (Fig. 6 & others in Nature article)Diagram Diagram (simplified - see article in webpage)Diagram Animation - S1 movie (based on X-ray structure, assays of conformational changes & molecular dynamics simulations)Animation

21.  Many channels have multiple open &/or closed states. There may be an inactivated state, as in the hypothetical example above.  Voltage-gated K + channels undergo transient inactivation after opening. In the inactivated state, the channel cannot open even if the voltage is favorable. This results in a time delay before the channel can reopen. Inactivation:

22. The N-terminus of the Shaker channel (or part of a separate subunit in some voltage-activated channels) is essential for inactivation. Mutants that lack this domain do not inactivate. Adding back a peptide equivalent to this domain restores the ability to inactivate.

23. See a more detailed diagram (Fig. 3).diagram In some voltage-gated K + channels, entrance of the N-terminus into the channel is followed by a conformational change in the selectivity filter that contributes to the process of inactivation. A "ball & chain" mechanism of inactivation has been postulated, in which the N-terminus of one of the 4 copies of the channel protein enters the channel from the cytosolic side of the membrane to inhibit ion flow.