Channels and pumps
Pumps:
Passive and active transports
Active: Uniport, symport and antiport
Energy sources for active transport: gradient, ATP and light
Mechanism for transport: 4 stages for P-type ATPase
Channels:
Gap junction and bystander effect.

Models of Pumps
P-type ATPase pumps:
Ca 2+ pump
Na + -K + pump
Co-transporters:
Na + -glucose pump
Na + - Ca 2+ exchange

Free energy across a membrane
D G = RT ln(c
2
/c
1
=2.303
RT log
) + ZF D V
10
(c
2
/c
1
) + ZF D V
Z: electrical charge of the passing molecule
F: faraday (23.1 kcal/V•mol )
R: gas constant (2 cal/mol•K)
T: absolute temperature (K)
D V: membrane potential

Special channels
Ligand (transmitter)-gated channels
Mechanism for activation and desensitization: acetylcholine binds to the receptor and induces a conformational change.
Voltage-gated channels
Mechanism for operation: actin potential, depolarization and repolarization.
Equilibrium potential ( D Veq)
Measurement of membrane conduct:
Patch-clamp techniques
Isolation of receptors using nature receptor-binding molecules.

Structures of the ion channels
Structure similarity among K + , Na + and Ca 2+ channels.
Mechanism for ion selection:
Energetic based selection (K + channel)
Size exclusive selection (Na + channel).
The role of amino acid residues of the selectivity filter.
Models for ion channels:
Two-site model
Ball and chain model.
Using trypsin and mutagenesis to study the mechanism of ion channels.

Natural acetylcholine receptor binding molecules
Nicotine Cobratoxin
Zeng and Hawrot, J. Biol. Chem, 2002

Isolation of acetylcholine receptors using nature resources
Cell extract from torpedo
Bungarotoxin or
Corbratoxin
Elute bound proteins
Selection of snake toxin binding molecule

Coordination of ion channels

Fugu (puffer fish): A fish to die for
Tetrodotoxin (from puffer fish) is
270 times more toxic than cyanide.

Clinical importance of membrane pumps and channels
Cardiovascular disease:
Digitalis, a drug for congestive heart failure
Caner:
MDR - multidrug resistance protein
Gap junction and gene therapy for cancer
Cystic fibrosis (CF):
Cystic fibrosis transmembrane regulator (CFTR)

Electrical activation in the 3D heart model
QuickTime™ and a
YUV420 codec decompressor are needed to see this picture.
QuickTime™ and a
YUV420 codec decompressor are needed to see this picture.
Reproduced with permission of Physiome Sciences, Inc., 307 College
Road East, Princeton, New Jersey

MDR and Cancer
About 40% of cancer developed multiple drug resistance due to amplification of a part of the genome contains Mdr1 .

Gap junctions and bystander effect
Griffin GD, Oak Ridge National Laboratory
Subauste MC, et al, J. Biol. Chem., 2001

CFTR coordinates Cl , Na + and water transport

Sound induced signaling pathway

Touching
Pressure
Temperature
Hot pepper

CREB won the Nobel Prize
2000 Nobel Prize for Physiology or Medicine was awarded to three prominent scientists:
Arvid Carlsson, Paul Greengard and Eric Kandel
Dr. Kandel, Columbia University, found that for the long-term memory to occur, certain genes needed to be “turned on” or activated through the release of a protein known as CREB 1.

Aplysia (boasts large neurons) - a tool to study learning and memory
Short-term memories, which are stored for minutes to hours, rely on the phosphorylation of certain ion channels. This increases calcium flows and thereby indirectly promotes the transfer of neurotransmitters, thus reinforcing the protective reflex.
Long-term memory can persist for weeks and relies on more widespread changes affecting the entire cell. Altered gene expression patterns mean that new proteins are produced, which can permanently affect the shape, size and sensitivity of the synapse.
Dr. Kandel’s Lab
How molecular changes in a synapse may produce short-term memory and longterm memory in the sea slug, Aplysia

GH signaling pathway

Mouse teeth and Nobel Prize
The Nobel Prize in Physiology or Medicine 1986
Dr. Stanley Cohen, Vanderbilt University School of
Medicine Nashville, TN, USA
Dr. Rita Levi-Montalcini, Institute of Cell Biology of the C.N.R Rome, Italy

Regulation of MAP kinase pathways

Major signaling pathways relevant to cancer in human

Molecular biology methods for studying signal transduction
Know ligand binding domain
Known
RTK
Sequencing database search
Unknown
RTK
Dominant negative mutant
Ligand binding domain
Affinity column for ligand purification

Signal transduction and diseases
Src
• Structure: two SH domains and one kinase domain with Tyr phosphorylation site on tail (Y527).
• Mechanism for activation: three ways of activation.
• V-Sar vs. c-Src: Tail or no tail, that is the problem.
Cholera
• Lock GTP in G a s and keep it active.
Whooping cough
• Lock GDP in G a i and keep it inactive.

Nobel price for studying Rous sarcoma virus
Peyton Rous, Rockefeller University, 1966 Nobel Laureate in
Medicine .
In 1910, Dr. Rous found that sarcomas in hens could be transmitted to fowl of the same inbred stock not only by grafting tumor cells but also by injecting a submicroscopic agent extractable from them; this discovery gave rise to the virus theory of cancer causation.

v-Src vs. c-
Src
Tyr 527

V. Cholera in action

Whooping cough

Light induced signaling
TRP-transient receptor potential family: votagegated K + or cNTP gated channel
INAD complex with 5 PDZ
(postsynaptic density protein) domains

Olfactory signaling pathways

Bitter and sweet tastes induced signaling pathways
Bitter signaling Sweet signaling

TM
TM

CML Diagnosis - Bone marrow
An abnormality of chromosomes:
The presence of the Philadelphia chromosome (Ph), a shortened chromosome number 22, in the marrow cells.
95% of CML cases are associated with a particular chromosomal translocation [t(9;22)], which creates a new gene bcr-abl .
Philadelphia chromosome

FISHing the Philadelphia
Multi color FISH with three different fluorochromes.
Chromosome painting probes are labeled in three fluorochromes which would display seven different colors.
The paints are hybridized to chromosomes from a chronic myeloic leukemia
(CML) cell line showing various chromosome rearrangements.

Brc and Abl genes

Gleevec TM binds to the tyrosine kinase domain of the Abl protein

Milestones for the discovery of Gleevec TM
Cancer Res.
1996 Jan 1;56(1):100-4.
Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2phenylaminopyrimidine derivative.
Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Druker BJ, Lydon NB.
Nat Med.
1996 May;2(5):561-6.
Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon
NB.
N Engl J Med.
2001 Apr 5;344(14):1031-7.
Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.
Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H,
Capdeville R, Ohno-Jones S, Sawyers CL.
N Engl J Med.
2001 Apr 5;344(14):1038-42.
Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.
Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, Capdeville R, Talpaz M.