Membrane Channels and Pumps

1. Mechanism and Regulation

2. Clinical and Physiological Relevance

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:

Ca2+ pump

Na+-K+ pump

Co-transporters:

Na+-glucose pump

Na+- Ca2+ exchange

G = RT ln(c2/c1) + ZFV =2.303RT log10 (c2/c1) + ZFV

Z: electrical charge of the passing moleculeF: faraday (23.1 kcal/V•mol )R: gas constant (2 cal/mol•K)T: absolute temperature (K)V: membrane potential

Free energy across a membrane

Specific Channels

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 (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 Ca2+ 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

Selection of snake toxin

binding molecule

Elute bound proteins

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 decompressorare 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

Subauste MC, et al, J. Biol. Chem., 2001

Griffin GD, Oak Ridge National Laboratory

CFTR coordinates Cl-, Na+ and water transport

Sound induced signaling pathway

Touching

Pressure

Temperature

Hot pepper

Signal Transduction

1. 7TM, GH and RTK Signaling Pathways

2. Clinical and Physiological Relevance

CREB won the Nobel Prize

2000 Nobel Prize for Physiology or Medicinewas 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

How molecular changes in a synapse may produce short-term memory and long-term memory in the sea slug, Aplysia

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

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

Unknown RTK

Know ligand binding domain

Dominant negative mutant

Ligand binding domain

Affinity column for ligand

purification

Sequencing

database search

Known RTK

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 Gs and keep it active.

Whooping cough

• Lock GDP in Gi 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: votage-gated 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

Development of GleevecTM (STI-571)

1. Identifying Target Gene

2. Developing GleevecTM

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

GleevecTM binds to the tyrosine kinase domain of the Abl protein

Milestones for the discovery of GleevecTM

Cancer Res. 1996 Jan 1;56(1):100-4. Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine 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.