The diffusion of ions across cell membranes through protein channels is essential to many biological processes. Membrane-embedded potassium channels transiently open in response to particular stimuli, thereby furnishing a specialized tunnel through which K+ ions flow down a concentration gradient. In the case of eukaryotic nerve cells, for example, potassium channels play a central role in restoring a negative membrane potential after sodium ions have entered the cell through transiently opened sodium channels and depolarized the membrane. Since K+ concentrations at rest are higher inside the cell than out, K+ ions leave the cell when potassium channels open in response to the sodium influx-induced depolarization. Nerve cell potassium channels thus shape the action potential through a voltage sensitive gating mechanism.
Voltage gated potassium channels are members of a large protein family found in all of life's kingdoms. They show significant sequence and structural homology, especially in the pore domain, which provides the structural conduit for ion conductance. Several features of voltage gated potassium channels are especially remarkable. First, the channels are able to discriminate between ions, providing high flux rates for K+ in their open state, while essentially remaining impermeant to Na+, even though the latter is a smaller ion. Second, although the discrimination just mentioned would seem to indicate solid, specific interactions with K+ that might be expected to slow conductance, the channel throughput rate of these ions approaches that limited by diffusion. Third, these channels respond to membrane electrical potential changes by conformational alterations that either open or close the channel. The structural bases for these various functions have been determined for a variety of potassium channels, and are the subject of this tutorial.
II. Ion Conductance and Selectivity in a Prokaryotic K+Channel
To the left is the membrane spanning region of the K+ channel from Streptomyces lividans, KcsA (Doyle, et al., 1998). The channel has been crystalized in its open state, with K+ ions in its pore.
Like all members of the K+ channel family, the KcsA protein is a homotetramer. The structure is oriented with its extracellular region facing up, its intracellular region at bottom. In this view, the structure has been likened to an inverted teepee.
Each of the four monomers comprises a selectivity filter, an inner helix, an outer helix, a pore helix, and a turret loop. The selectivity filter contains the highly conserved potassium channel signature sequence (for KcsA, TVGYG). Mutations of these residues impair ion discrimination (see below).
The assembly of monomers into the tetramer thus provides a central channel through which K+ ions can flow, with its narrowest diameter at the junction of the selectivity filters.
KcsA has been modeled in the planes of a hypothetical membrane. Of note are the rings of aromatic amino acid sidechains on the membrane-facing surfaces, provided by both the inner and outer helices of each monomer. The structural elements of the transmembrane region contain mostly hydrophobic residues, which, except for the selectivity filter (see below), create a non-polar cavity through which K+ ions move. Regions with charged amino acids are primarily in the extracellular and intracellular domains.
We can now turn to the features of the KcsA channel that provide a pathway for rapid and selective K+ conductance. In the following, flow is visualized as outward, from cytosol (down) to extracellular space (up). Flow begins at the intracellular entryway, continues into a cavity of variable width (10 Angstroms at its widest), and proceeds through the selectivity filter, exiting the membrane at the extracellular entryway.
III. Voltage Gating in a Eukaryotic K+ Channel
A mammalian voltage dependent K+ channel of the Shaker family is displayed at left. The voltage sensor and pore domains, the T1 domain, and an associated, regulatory oxido-reductase ß subunit are indicated.
Focusing on the transmembrane domains, it can be seen that Shaker is a homotetramer, as is KcsA (above). As above, the structure is oriented with the extracellular face on top and intracellular face at bottom.
Each monomer comprises voltage sensor and pore domains, connected by a linker helix.
Like its prokaryotic counterpart, the pore domain contains an inner helix (S6), an outer helix (S5), a pore helix, a turret loop, and a selectivity filter. The voltage sensor contains 4 helices: , S1, S2, S3, and S4. The S4-S5 linker helix joins the two domains by connecting S4 and S5.
In order to visualize the mechanics of voltage gating, we need to consider the dispositions of several key helices of the structure, treated in the next two steps.
Helix S4 of the voltage sensor contains 4 essential arginine residues (gating charge residues) that change position in order to open or close the channel. In the structure shown, the channel is open with the gating charge arg's positioned near the top (extracellular) side of the molecule. In vivo, this channel state would occur in response to Na+ influx depolarizing the membrane at the start of an action potential. As the membrane potential is restored to a negative state by the outward flow of potassium ions through the open channel, the positively charged gating arg's would be be drawn closer to the intracellular side of the membrane (down in the view at left), thus closing the channel by driving a conformational change of the protein (see next step).
The S4-S5 linker helix interacts with the inner helix (S6) of the pore domain, the latter curving and running parallel to the horizontal plane of the membrane (not shown) and serving as a platform-like "receptor" for the S4-S5 linker helix above it. Thus, changes in the disposition of the S4 helix driven by shifts of the positively charged gating arg's change the disposition of the S4-S5 linker helix and this in turn affects the disposition of the S6 helix, opening or closing the channel depending on the position of the gating arg's .
Now the ability of the positively charged gating arg's to regulate channel opening and closing in response to membrane potential can be visualized (see Figure 1). In a resting cell, the membrane potential is negative (inside of cell is negative relative to outside). The positively charged gating charge residues lie closer to the intracellular side of the channel, and the channel is closed. As Na+ enters the cell at the begining of an action potential, the membrane is depolarized, the gating charge residues shift toward the extracellular side of the channel, and the channel opens, allowing K+ to flow out of the cell. As the resting membrane potential is thus restored, the gating charge residues shift back toward the intracellular side of the channel, and the channel closes.
In the tetrameric channel, the voltage sensor domains can be seen at the four corners of the square-shaped structure formed by the assembly of the pore domains. As already discussed, the voltage sensor domains connect to the pore domains through the S4-S5 linker helix. The blinking helices reflect the order of the "chain reaction" of the conformational alterations of S4, S4-S5, and S6 in response to gating charge arginine shifts. As mentioned, the structure at left is in the open state with K+ ions in the channel pore.
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Long SB, Campbell EB, Mackinnon R.. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309(5736):897-903. (2005).
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 414(6859):43-8. (2001).
Zhou Y, MacKinnon R.. The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates. J Mol Biol. 333(5):965-75. (2003).
Acknowledgement: The format of this web page is modified from a template provided by Dr. Angel Herraez, Bioquimica y Biologia Molecular, Universidad de Alcala,
E-28871, Alcala de Henares (Madrid), Spain.