Understanding Transport through Membranes

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Understanding Transport through Membranes
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The importance of ion transport through membranes
Water is an electrically polarizable substance,
which means that its molecules rearrange in an
ions 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
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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

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Ion Channels Have Very High Turnover Ratios
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
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What do we know based on molecular biology?
Linear Sequence
Proposed topology
Membrane Propensity
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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)

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Structure-Function Relations in a
Voltage-Dependent Channel
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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

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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
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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

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Crystal Structure of the Streptomyces K Channel
Doyle et al. 1998

• 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

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Entryway

• Entryways to the channel have several negatively
  charged amino acid residues which increase the
  local concentration of cations (K and Na)

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Understanding Permeation and Selectivity

• 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

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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

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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)

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Selectivity FilterHow does K channel
distinguish K from Na?

• Located in narrow region of the channel
• Contains Gly-Tyr-Gly AA residues
• Forces K to lose its 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

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Selectivity FilterHow 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

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The Chloride Channel breaks the Rules!
ClC single channel behavior suggests a double
barrel arrangement
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Anionic Selectivity Appears to be Based on Ion
Stabilization by Helix Dipoles
Cl- coordination site
Cl Channel
K Channel
Channel entry