Last Class:
A. Posttranscriptional Regulations
1. Alternative RNA Splicing; 2. 3’ cleavage; 3. RNA editing; 4. Nucleus
transportation; 5. Localization of RNA at the cytoplasm; 6. Translational
initiation, SiRNA
B. Plasma Membrane
1. Lipid bilayer, 2. lipid molecules, 3. plasma membrane structures, 4.
lipid molecules as signals, 5. general idea of membrane proteins
Detergent to solubilize and purify membrane proteins
A detergent micelle
Solubilize
membrane proteins
with detergent
Different Detergents
Ionic (strong) or nonionic (weak)
Mild detergent for the
solubilizing, purification,
and reconstitution of
membrane protein
functions to study the
functions of membrane
proteins in simplified
environment
The study of membrane proteins
Most prominent example
red blood cells
No nucleus or internal organelles
The preparation of red blood cell membranes
15 major membrane proteins in
red blood cells
Label them with impermeable
dyes can determine the
location on layers
Membrane proteins
are diffusible
Techniques to study protein motion on membrane
(Fluorescence Recovery After Photobleaching
(Fluorescence Loss In Photobleaching)
Proteins restrictions
Tight Junction is one kind of them
Proteins and lipids on the outer layers can’t move
to other compartments
Protein distributions in a
guinea pig sperm cell
4 ways of protein restrictions
1. self-assembly
2. Tethered to macromolecules outside
3. Tethered to macromolecules inside
4. Cell-cell adhesion
Carbohydrate layer (Glycocalyx) on the cell surface
Protecting the cell surface from mechanical and
chemical damage
Lymphocyte stained with ruthenium red
Diagram of glycocalyx
Summary
•membrane proteins and their anchoring
models
•Methods to study membrane proteins,
detergents
•diffusion, distribution, methods to study
protein motion and distribution
•glycocalyx, proteoglycan
• Membrane Transport of Small Molecules
and the Electrical Properties of
Membranes
Permeability of plasma
membrane
General principles I
Permeability of plasma
membrane
General principles II
Permeability coefficient
(cm/sec)
Membrane Transport Proteins
Carrier Protein and Channel Protein
Transportation Models
Passive and Active Transport
Electrochemical and concentration gradient, membrane potential
Carrier proteins: passive and active
Channels: always passive
Electrochemical Gradient
Is the combinatory effect of concentration gradient
and membrane potentials
Ionophores can serve as channels and carriers for ions
Example: A23187, calcium permeabilizing agent
Carrier Proteins and Active Membrane Transportation
Conformational change of a carrier protein
Mediates passive transport
Change is spontaneous and random, so
dependent on concentration
Kinetics of simple and carrier-mediated diffusions
3 ways of driving active transportation utilizing
passive carriers
1. Coupled carriers
2. ATP-driven pumps
3. Light-driven pumps
3 types of carrier-mediated transport
Coupled carriers
Coupled transportation of
glucose and Na+
Cooperative binding of Na+
and glucose to the carrier.
Outer surface, Na+ high
concentration induces the
high affinity of glucose to
carrier
Transcellular transport
Tight junction separates apical and basal/lateral spaces
Apical: glucose and Na+ coupling; basal/lateral: glucose is passive, Na+
maintained by ATP-driven pump
Na+-K+ Pump, ATPase
P-type transport ATPase (dependent on phosphorylation)
Cycles of Na+-K+ Pump
Calcium Pump
ATP binding and hydrolysis can push calcium inside by bring
N and P domain together
A typical Ion Channel
1. selectivity, 2. Gated (close and open)
The gating of Ion Channels
The Structure of bacterial K+ channel
Selectivity 10,000 fold over Na, although K+ 0.133nm,
Na+ 0.095 nm
The Selectivity of bacterial K+ channel
Carbonyl oxygens at selective filter
Gating Model of K+ channel
Selectivity filter is fixed, the vestibule open and close
like a diaphragm
Summary
• Membrane transportation, carrier protein,
channel protein
• Active transportation, passive transportation
• Carrier Proteins, coupled carriers, ATPases,
Na+-K+ Pump
• Gating mechanisms of Ion Channels, K+
channel selectivity
• Cell Communication
•
•
•
•
General Concepts
Ion channel-linked receptors
*GPCR signaling
*Enzyme linked receptors
•General Concepts
A simple intracellular
signaling pathway
Extracellular
signaling molecules
bind to receptors
Signals can be tranmitted either short or long
distances (I)
Signals can be tranmitted either short or long
distances (II)
For Long distance, two
typical ways
Endocrine signaling
Different cells need specific
ligands and receptors
Synaptic signaling
More efficient, same set of
ligands and receptors
Signaling via gap junctions
No ligand-receptor system needed
Combinatory
effect of multiple
inputs
Different receptor type and intracellular signaling
molecules determine the ultimate response
Many signaling molecules have short lifetime
NO (nitric oxide) induces the relaxation of SMC
The function of viagra is to inhibit cyclic GMP phosphodiesterase, hence
elongate the lifetime of cyclic GMP and relaxation
2 steps of responses may occur upon stimulation
Secon
Cell Surface receptors belong to three classes: 1. ion-channel-linked
receptors, 2. G-protein-linked receptors, 3. enzyme-linked receptors
Cell Surface receptors belong to three classes: 1. ion-channel-linked
receptors, 2. G-protein-linked receptors, 3. enzyme-linked receptors
Different Kinds of intracellular
proteins serving as signaling
molecules
1. Relay proteins
2. Messenger proteins
3. Adaptor proteins
4. Amplifier proteins
5. Transducer proteins
6. Bifurcation proteins
7. Integrator proteins
8. Latent gene regulatory proteins
Two kinds of molecule switch events
Phosphorylation and GTP binding
Signaling integration
Intracellular signaling complexes enhance the speed, efficiency, and
specificity
2 types: Preassembled vs. Assembled after stimulation
Intracellular signaling complexes enhance the speed, efficiency, and
specificity
2 types: Preassembled vs. Assembled after stimulation
Binding domains for interactions between proteins and complex assembly
Cells can be sensitive to
subtle difference in
environment
1. Multiple ligands are
needed for one
signaling molecule
Cells can be sensitive to subtle difference in
environment
2. Multiple ligated molecules are needed to be
assembled to be functional
Cells can be sensitive to subtle
difference in environment
3. Positive feedback can enhance the
response drastically
Cells can adjust their sensitivity to stimuli by desensitization process
Summary
• Typical signaling transduction pathway: 1. ligandreceptor, 2. gap junctions
• Different inputs, receptors, intracellular signaling
network determine the ultimate response
• The importance of lifetime of molecules
• Different steps of responses
• The types of receptors: ion-channel-linked, G
protein coupled, enzyme-linked receptors
• Intracellular signaling molecules, signaling switches,
signaling integration, signaling complex assembly,
protein-protein binding modulus, signaling
amplification, signaling desensitization