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 ChannelsThree 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

Carrier Substrate Turnover (s-1)

Valinomycin 3 x 104 Na-K-ATPase 5 x 102

Ca-ATPase 2 x 102 Glucose

transporter 0.1-1.3 x 104

Channel Substrate Turnover (s-1)

Na-channel (V) 7 x 106 Ca-channel (V) 1.9 x 106 K-channel (Ca,

V) 0.2-3 x 107

ACh receptor 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

Ion Channels Have Very High Turnover Ratios

Very few ions are needed to generate a sizable transmembrane potential in cells

Also …,

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)

P loop

VoltageSensing Slow

Inactivation

SelectiveOligomerization

FastInactivation

SelectivityPermeation

&

Gating

Structure-Function Relations in a Voltage-Dependent Channel

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

• Maintains membrane potential• Regulates cell volume• Modulates electrical excitability of neurons

Role of K+ Channel

Residues that interact with scorpion toxin Residues that interact

with tetraethylammonium

Residues that interact with K+ ions

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

TM1

P-loop

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

Doyle et al. 1998

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

W+

__

+ 1

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 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 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 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

The Chloride Channel breaks the Rules!

The structure of the ClC chloride channel deviates from “classical” membrane protein architectures

Two-fold symmetry

Helix packing is very complex

ClC single channel behavior suggests a double barrel arrangement:

Cl Channel

K Channel

Anionic Selectivity Appears to be Based on Ion Stabilization by Helix Dipoles

Cl- coordination site

Channel entry