S. lividans pH-Gated
Potassium Channel
Hardy Evans '15 and Kevin Pan '15
Contents:
I. Introduction
Potassium channels allow potassium ions
to diffuse rapidly across cell membranes. This diffusion plays an
important role in numerous biological processes, including signal
transduction, cardiac automaticity, and homeostasis. Potassium
channels are tasked with combining incredibly rapid conduction
rates--on the order of 10^8 ions per second--with nearly flawless
selectivity. Because nearly all potassium channels face this exact
challenge, their structure is highly conserved amongst different
organisms and cell types. KCSA is an integral membrane protein from Streptomyces
lividans with amino acid sequence similarity to all known K+
channels. Like all potassium channels, KCSA possesses highly specific
architecture, particularly in its selectivity filter, the component
responsible for distinguishing ligand specificity. The structural
properties of KCSA are critical to its function, as they manipulate
the thermodynamic and kinetics of transmembrane ion flux in a way that
allows cooperation between the normally contradictory concepts of
speed and accuracy.
II. General Structure
KCSA is a tetramer with four membrane-spanning
. Each subunit contains two transmembrane
, adjoined by the roughly 30 amino acid pore region. The pore region
consists of the
. The protein has four-fold symmetry about the pore. Each subunit
enters the tetramer such that one transmembrane helix faces the
central pore, while two outer helices face the lipid membrane. The
inner helices bend slightly outwards from the pore, at an angle of
25 degrees to the membrane normal. These inner helices house the
extracellular region of the pore, where the potassium selectivity
filter is located. The four inner helices pack tightly against each
other near the intracellular region of the protein, resembling an
inverted teepee. The pore helices are inserted between the "poles"
of the teepee, with the carboxy-termini pointing into the center of
the channel. This particular arrangement provides multiple
inter-subunit contacts, which help hold the four subunits together.
III. pH-gating
Crystallography of KCSA in the closed conformation shows a
crossing of the cytosolic ends of the trans-membrane helices,
blocking the pore region. During channel opening, this crossing
widens in order to allow access to the pore. This widening is
mediated by proton-dependent disruption of subunit interactions. At
neutral pH, Glu118,
Arg117 and His25
interact to create an
inter- and intra-subunit network of salt bridges and hydrogen
bonds
. These interactions constrain the ends of the trans-membrane helices
at the bundle crossing, stabilizing the closed state. At acidic pH,
the glutamates are protonated and become neutral, breaking their
interactions with arginine and histidine. and increasing the net
positive charge on the trans-membrane helices. This destabilizes the
bundle-crossing via electrostatic repulsion between the
aforementioned residues. This causes the trans-membrane helices to
separate, opening
the channel.
IV. Selectivity Filter
The selectivity filter is of a K+ channel is highly
conserved amongst all species and cell types. The selectivity
filter consists of the amino acid sequence TVGYG
at
, which is conserved in all K+ channels. The Val and Tyr side chains
from the amino acid sequence point away from the pore and make
specific interactions with the tilted pore helix. The four Tyr78
side chains combine with the Trp67
and 68 residues from the
pore helix to form a massive sheet of
that is positioned around the selectivity filter. There are four
strands of sequence, contributed by each of the subunits that are
arranged with their carbonyl oxygen atoms pointed inward toward
the ion conduction pathway. The selectivity filter can undergo a
transition involving two amide planes between
and
. Potassium ions diffuse through the channel at rates of approaching 10^8
ions/second under certain conditions. In order to
catalyze these high diffusion rates, a K+ ion must dehydrate and
cross the selectivity filter within 10 ns.
There are four binding sites in the central pore. Each binding site
is a cage-structure formed by eight oxygen
atoms on the vertices of a cube, each provided by the C-terminal end
of a pore helix directed into
the binding
. These ends are associated with a significant negative end charge, due
to carbonyl groups that do not form any structural interactions with
the rest of the protein. These oxygen atoms "hydrate" the K+
ion, stabilizing the transition from the aqueous extracellular
environment to the relatively hydrophobic intramembrane environment.
Fascinatingly, the distance between the K+ ion and each oxygen is
exactly equivalent to the distance between a hydrated K+ ion and a
water molecule, which allows easy de-solvation of the ion. The size
of each oxygen cage is such that sodium
is
to interact with the carbonyl oxygens, thus providing
selectivity.
In order to facilitate high conduction rates, the binding of K+
ions causes the molecule to go from a "collapsed", or "pinched",
conformation to a relatively unstable conductive conformation.. The dedication of binding energy to change
conformation reduces K+ affinity, increasing rate of dissociation
and thus conduction. Additionally, the binding of multiple cations
leads to electrostatic repulsion, which also decreases affinity and
increases conduction.
V. References
1. Doyle DA, Cabral JM, Pfuetzner RA, Kuo A, Gulbis JM, et al. (1998)
The Structure of the Potassium Channel: Molecular Basis of K+
Conduction and Selectivity.
2. Thompson AN, Posson DJ, Parsa PV, Nimigean CM (2008) Molecular
mechanism of pH sensing in KcsA potassium channels.
3. Berneche S, Roux B (2005) A gate in the selectivity filter of
potassium channels. Structure 13: 591-600.
4. Blunck R, Cordero-Morales JF, Cuello LG, Perozo E, Bezanilla F
(2006) Detection of the opening of the bundle crossing in KcsA with
fluorescence lifetime spectroscopy reveals the existence of two gates
for ion conduction. J Gen Physiol 128: 569-581.
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