Eduardo Perozo

1
Lecture 2
Ion Channel Biochemistry and Structure

• Eduardo Perozo
• 4-45 Jordan Hall
• 243-6580
• eperozo_at_virginia.edu

2
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
3
What do we know based on molecular biology?
Linear Sequence
Proposed topology
Membrane Propensity
4
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)

5
Voltage-Dependent Channels
6
Ligand-Gated Channels
7
Other Channel Families
8
Structure-Function Relations in a
Voltage-Dependent Channel
9
Several ways to generate Diversity
Gene variability
Gene A
Gene B
Gene C
10

Diversity in K channels
11
Actual Experimental Results
12
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

13
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

14
3D Single particle reconstruction of Shaker
Grigorieff et al 2001
15
The Crystal Structure of KvAP

• KvAP structure
• Obtained from an hyperthermophile organism
• Determined with the aid of Fab fragments
• S4 appears to be intracellular, even outside
  the membrane

16
Evaluating current KvAP structural models
The Paddle Model
A Recent Canonical Model
S3
S2
S1
S4
Jiang et al 2003b
Roux et al, unpublished
17
Mechanistic Models of Voltage Sensing
The Canonical Model
The Paddle Model
The Moving Field Model
18
Structure of the Torpedo Acetylcholine Receptor
Unwin, 1993, 94, 96, 2000
2a, b, g, d/e




There are 5 homologous subunits


FT
Each subunit has four putative TM segments



There is a large aqueous vestibule (extracellular)



Single Subunit
19
Structure of the Torpedo Acetylcholine Receptor
(4.5 Å)
ACh, Bungarotoxin Binding
TM segments, Gate
Cytoskeletal Anchor?
20

Operation of the ACh Receptor Channel
Conformational changes in the TM segments of the
AChR
21
Structure of the Acetylcholine Binding Protein
(4.5 Å)
22
The putative Acetylcholine Binding site
p-cation interactions play a key role
HEPES at 50 occupancy?
23
The Chloride Channel breaks the Rules!
ClC single channel behavior suggests a double
barrel arrangement
24
Anionic Selectivity Appears to be Based on Ion
Stabilization by Helix Dipoles
Cl- coordination site
Cl Channel
K Channel
Channel entry