Contents of this page:
Introduction to voltage-gated K+ channels
Gating & inactivation
Note: Rather than try to cover the many different types of membrane channels, this class will focus on K+ channels whose structures have been solved at atomic resolution. Channels discussed elsewhere in this web include porins, gramicidin, and endoplasmic reticulum calcium-release channels.
|Voltage-gated K+ channels mediate outward K+ currents during nerve
action potentials. Important advances in understanding have come from:
Four identical copies of the K+ channel protein, arranged as a ring, form the walls of a K+ channel.
Hydropathy analysis and topology studies predicted the presence of
six transmembrane a-helices
in the voltage-gated K+ channel protein.
K+ channels are highly selective for K+, e.g., relative to Na+. The selectivity filter that determines which cation (e.g., Na+ or K+) 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.
The first K+ channel for which an X-ray crystallographic structure was solved is the KcsA K+ channel from Streptomyces lividans, depicted in cartoon display at right. In this channel protein there are only two transmembrane a-helices, corresponding to helices # 5 & 6 in the voltage-gated channel. An intervening segment includes the K+ channel consensus sequence.
The left diagram is a side view of the KcsA channel (normal to the membrane), while the other view looks down the channel from the extracellular side of the membrane.
|As K+ enters the channel, its waters of hydration are replaced
oxygen atoms lining the
Four binding sites for K+ within the selectivity filter are defined by 5 rings of oxygen atoms (see Chime exercise below). Each ring consists of 4 oxygen atoms, one from each subunit. Most are backbone oxygen atoms of the consensus sequence. Each bound K+ interacts with eight oxygen atoms, as it sits between two of the rings of oxygen atoms.
At right, a K+ ion (colored pink) is seen closely interacting with the outermost ring of oxygen atoms (colored red) of the selectivity filter.
For additional images see a webpage describing work of R. MacKinnon maintained by the Howard Hughes Medical Institute, plus figures p. 736-737 of Biochemistry, 3rd Ed., Voet & Voet.
Solved X-ray structures such as that depicted above, which average over many copies of the channel in the crystal, show K+ in all 4 binding sites. Based on predicted electrostatic repulsion and other evidence, K+ is assumed to occupy actually every other binding site, with a water molecule in each intervening site. K+ ions and water molecules 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. The arrangement of oxygen atoms surrounding each K+ ion within the selectivity filter mimics that of a hydrated K+ ion. Thus the energy barrier for entry and exit of K+ ions is low.
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, the presence of
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 collapsed state.
Explore at right the structure of the bacterial KcsA channel.
Bacterial K+ Channel
Channel gating: The structure of a Ca++-dependent K+ channel from Methanobacterium thermoautrophicum, designated 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 at right.
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 (above), the outward splaying of bent helices provides a large opening at the cytosolic end of the open MthK channel (side view in diagram at near right).
|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. Instead it induces the channel conformational changes responsible for gating.|
Explore at right the open conformation of the bacterial MthK channel and its regulatory gating ring.
Ca++-Gated K+ Channel
Mutational analysis first established that
positively charged residues in helix #4
are essential for voltage gating. In helix #4, every 3rd
residue is arginine or lysine, while 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 positive charges effectively move outward.
|The core of both voltage-gated
channels (selectivity filter & two transmembrane a-helices
of each of four copies of the protein) is similar
to that of other K+ channels.
The bacterial KvAP channel, in the open conformation, is depicted at right.
Some a-helices of the voltage-sensing domain of the bacterial KvAP channel differ from the expected transmembrane orientation. The positively charged voltage-sensing helix #4, and part of what was earlier designated helix #3, associate in a helix-turn-helix to form a "paddle" shape at the periphery of the channel (colored magenta in the diagram above). For the channel structure depicted above, Fab fragments of monoclonal antibodies raised to the voltage-sensor domain of the channel protein were used to promote crystallization.
The structure of the mammalian equivalent of
channel has been determined in the absence of FAB
fragments but in complex with a regulatory protein. In this channel,
a-helices of the
voltage-sensing domain are tilted relative
to the plane of the membrane but have a
For diagrams see a website of the Brookhaven National Laboratory, and an article by Long et al. (requiring a subscription to Science).
Some differences between solved KvAP and Kv1.2 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.
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.
Many channels have multiple
open and/or closed states. There
may be an
inactivated state, as in the hypothetical example at right.
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.
|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.
A "ball & chain" mechanism
of inactivation has been postulated, in which the N-terminus of one
of the four copies of the channel proteins enters the channel from the
cytosolic side of the membrane to inhibit ion flow.
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.
Copyright © 1998-2008 by Joyce J. Diwan. All rights reserved.
Additional material on K+