Lecture 2

Membrane Structure & Ligand-gated channels

• In Memory of my good friend and colleague

• Massimo Sassaroli

Membrane StructureLearning Objectives

Know the major classes of natural lipids, their structural characteristics and the properties of head groups and hydrocarbon chains

Understand the thermodynamic basis of lipid and detergent assembly: the hydrophobic effect

Understand the physical connection between the shape of lipid molecules and that of the aggregates they form: critical packing parameter

Understand the connection between the configuration of acyl chains, their packing and the properties of the bilayer

Know the connection between bilayer structure and dynamics and translational diffusion of lipids and proteins. Experimental approach to the measurement of diffusion of membrane components: Fluorescence Recovery After Photobleaching

Understand the characteristics of the fluid phase bilayer as shown by diffraction experiments and computer simulations of lipid dynamics

Cell membranes are dynamic fluid structures. Most of their molecules are able to move about in the plane of the membrane

~30% of the proteins encoded in an animal cell’s genome are membrane proteins

Phosphatidyl Choline

Acyl Chains

Head Groups

Major classes of lipids

Glycolipids

A2: cleave at C2e.g. AA to PG and LT

A1: cleave at C1

C: cleave at C3 e.g. PIP2 to DAG and IP3

D: cleave at C3 e.g. phosphatidic acid

Phosphatidyl inositol phosphates

(C)

G = H T S

Hydrophobicity and thermodynamics of lipid assembly

•van der Waals force•

•electrostatic force

•hydrophobic force

Electrostatic interactions are lost when H2O binds to the

hydrocarbon chain. This translates to both an enthalpic and entropic (immobilization of

H2O) energy cost. Tight packing of hydrocarbon chains and

release of H2O back to the balk reduces G => Hydrophobic

effect

Saturated chains in the all-trans configuration, 19Å2

PE S39Å2

PC S50Å2

Membrane Proteins and Lipid-Protein Interactions

Learning Objectives

Modes of protein-membrane interaction.

Prediction of membrane protein structure and topology: Hydropathy analysis and the

‘positive‑inside’ rule.

Lipid‑modifications of proteins: hydrophobicity and membrane-binding affinity

Modulation of reversible protein-membrane binding: the myristoyl switch.

Phosphoinositide control of Ion Channel Activity

Monitoring PIP2 hydrolysis – translocation of PH-GFP

Keselman and Logothetis, (2007) Channels 1:113-123

i/o

Kir2.1

5 minPI(4,5)P

2

AASt PI(4,5)P2

O

O

O

O

H

OPO

O

O

OH

HO

OH

O

OPO

HO

O

PO

HOO

4 32

16

5

Kir channels run down upon channel excision and are reactivated by PIP2

0.1 1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

Kir2.3

Kir2.1

I/Im

ax

[diC8 PIP

2] (M)

AASt PIP2

Mg2+

M diC8101252.525

Ba

Ba

ACh

Kir2.3

ACh

Kir2.1

Modulation of Kir2 activity by PLC stimulation

diC8 PI(4,5)P2

O

O

O

O

H

OPO

O

O

OH

HO

OH

O

OPO

HO

O

PO

HOO

4 32

16

5

Channel PIP2-sensitive sites are congregated near helix bundle crossing and

H-I loop gates

PIP2 tethers the N- and C-termini to the inner leaflet of the plasma membrane gating the channel to the

open state

WORKING HYPOTHESIS

Kir3 modulation sites congregate around PIP2 sites

Cyan: PIP2 sites

Red: G sites

Pink: Na+ sites

Orange: Phosphorylation sites

1. Know the mechanism by which the metabolic state of the cell is coupled to membrane electrical events, such as those leading to secretion of insulin.

2. Know the mechanism of activation of G protein-gated K channels, as an example of a membrane-delimited pathway of regulating the activity of intracellular ligand-gated ion channels.

3. Modulation of Ion channels by soluble second messengers 4. Sensory transduction: Know the role of CNG channels in

phototransduction. Understand how the balance of CNG and K channels gives rise to the "dark current", which is inhibited during a light flash.

5. Know the subunit composition of nicotinic ACh channels and general topology of the subunits.

6. Know the general activation mechanism for NMDA and non-NMDA channels and role in LTP.

7. The Transient Receptor Potential (TRP) Channel Family

Intracellular and Extracellular Ligand-Gated Ion Channels

Learning Objectives

ATP-Sensitive K+ Channels

Electrostatic Channel-PIP2 Interactions

G Protein-sensitive K+ channels

Cytosolic domains of GIRK1

Nishida et al., 2002 Cell 111:957-65

Jin et al., 2002 Mol. Cell 10:469-481

Jin et al., Mol. Cell 10, 469 (2002)

G175 mutants of Kir3.4*

The cells of the Retina

The “dark” current and the response to light of a photoreceptor cell

Nicotinic ACh Receptor

Image reconstruction from cryo-EM

Two types of Glutamate Receptors

Glutamate Receptor ligand-binding domain