Transcript Document

Understanding Transport through Membranes
The importance of ion transport through membranes
Water is an electrically polarizable substance, which
means that its molecules rearrange in an ion’s electric
field, pointing negative oxygen atoms in the direction of
cations and positive hydrogen atoms towards anions.
These electrically stabilizing interactions are much
weaker in a less polarizable substance such as oil.
Thus, an ion will tend to stay in the water on either side
of a cell membrane rather than enter and cross the
membrane. Yet, numerous cellular processes ranging
from electrolyte transport across epithelia to electrical
signal production in neurons, depend on the flow of
ions across the membranes
Ion Channels
Three basic properties of ion channels:
• To conduct ions rapidly
• Exhibit high selectivity: only certain ion species flow while
others are excluded
• Conduction be regulated by processes known as gating,
i.e. ion conduction is turned on and off in response to
specific environmental stimuli
Ion Channels Have Very High Turnover Ratios
Carrier
Valinomycin
Na-K-ATPase
Ca-ATPase
Glucose
transporter
Channel
Na-channel (V)
Ca-channel (V)
K-channel (Ca,
V)
ACh receptor
Substrate Turnover
(s-1)
3 x 104
5 x 102
2 x 102
0.1-1.3 x 104
Substrate Turnover
(s-1)
7 x 106
1.9 x 106
0.2-3 x 107
2.3 x 107
As a comparison, the turnover ratio
(maximum number of processed
substrate molecules per active site,
per second) serves as a good
evidence for the physical concept of
pore. The turnover rates for some
known
carriers
or
active
transporters are compared to those
of several ion channels
Also …,
Very few ions are needed to
generate
a
sizable
transmembrane potential in cells
Unifying Themes in Ion Channel Structure
Polytopic Membrane Proteins
Oligomeric Arrangement
With Intrinsic Symmetry
Pore Size Correlates with the
Number of Subunits
•Voltage-Dependent
(Na+, K+, Ca++)
•Glutamate Receptors
•Ligand-Gated
(Ach, Gly, GABA,
5-HT)
•Mechanosensitive
•Connexins
(Gap Junctions)
Structure-Function Relations in a VoltageDependent Channel
Voltage
Sensing
Selectivity &
Permeation
Slow
Inactivation
P loop
Gating
Selective
Oligomerization
Fast
Inactivation
Introduction
• Membrane protein found in Streptomyces
lividans
• Analogous to K+ channels found in humans
• Selectively allows K+ ions to exit cells down their
concentration gradient
Role of K+ Channel
• Maintains membrane potential
• Regulates cell volume
• Modulates electrical excitability of neurons
Residues that
interact with K+ ions
Residues that
interact with
scorpion toxin
Residues that interact
with
tetraethylammonium
Pore loop proposed to reach
into the membrane and form
a selectivity filter
Structure
• Exists as a homo-tetramer
with 4 identical subunits
• Each subunit is comprised
of 3 alpha helices
• 2 helices are membrane
spanning
• 1 inner helix is responsible
for K+ selectivity
Crystal Structure of the Streptomyces K+ Channel
Doyle et al. 1998
P-loop
TM1
TM2
•KcsA is a homotetramer
•Each subunit contains two TM segments
•The selectivity filter is formed by an extended
structure positioned by a short tilted helix
Entryway
• Entryways to the
channel have several
negatively charged
amino acid residues
which increase the
local concentration of
cations (K+ and Na+)
Understanding Permeation and Selectivity
+
+
1
W
2
_ _
3
•K+ Ions are stabilized by backbone Carbonyls
•It is the matching of dehydration energies what determines selectivity
•High throughput is achieved by electrostatic repulsion between sites 1 and 2
Function of the Internal Pore
• Electrostatic barrier
to entry of K+ ion into
lipid bilayer overcome
by:
- Hydration of K+ ion
within membrane pore
-Stabilization provided
by short alpha helices
in the pore region of
each subunit w/
negatively charged
carboxyl termini
pointed at K+
How does
+
K
leave?
• 2 K+ ions at close proximity in
the filter propel each other
• This repulsion overcomes the
otherwise strong interaction
b/w ion and protein that allows
for rapid conduction
• Speed of conduction approaches
the theoretical limit of
unrestricted diffusion (108 ions/
second)
Selectivity Filter
How does K+ channel distinguish K+ from Na+?
 Located in narrow region of the
channel
 Contains Gly-Tyr-Gly AA residues
 Forces K+ to lose it’s hydrating water
molecules
 Carbonyl oxygen's in selectivity filter
stabilize K+ ions
 Aromatic amino acids line the filter
and act as springs to maintain
appropriate channel width for K+
 This favorable interaction with the
filter is not possible for Na+ because
Na+ is too small to make contact with
all the potential oxygen ligands of the
carbonyl termini of the short alpha
helices
Selectivity Filter
How does K+ channel distinguish K+ from Na+?
• Gly residues in the TVGYG sequence have dihedrals in or
near the left-handed helical region, allowing main chain
carbonyls point in one direction, towards the ions along the
pore.
• The oxygen atoms of the four sites surround K+ ions as water
molecules, paying for energetic costs of K+ dehydration
• Na+ ions too small for K+-sized binding site, so dehydration
energy is not compensated
The Chloride Channel breaks the Rules!
ClC single channel behavior suggests a double barrel arrangement:
The structure of the ClC chloride channel deviates from
“classical” membrane protein architectures
Helix packing is very complex
Two-fold symmetry
Anionic Selectivity Appears to be Based on Ion Stabilization by Helix Dipoles
Cl- coordination site
Cl Channel
K Channel
Channel entry