transport functions slide show 2014

8/12/2019 Transport Functions Slide Show 2014

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From the lecture missed on Feb. 5

Membrane Regulation: Main idea that students should know:

Functions of the ER

Quality control in the ER; ERAD: Unfolded protein response, Chaperones, Signals to

Nucleus, Proteosome.

Golgi apparatus: Structure, functions, vesicular transport, SNARES, ER/Golgi domains,

alterations of the lipid content, cargo sorting, roles of cholesterol and sphingolipids.,

Sorting signals for vesicles on the ER-Golgi-TGN pathway.

Lysosomes

Autophagosomes, Atg proteins.

Students should answer all of the questions posed in the Membrane Regulation PowerPoint

presentation.

Students should be able to discuss the origin and remediation of ER stress.

Students should be able to discuss the thermodynamic problems of vesicle-vesicle

fusion.

Students should be able to discuss how proteins may be attached to membranes and

how this attachment is regulated. This would involve Lectures 3 an and 4 as well as

lecture 5.


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Transport Functions/Properties of

Membranes

Transport across the plasma

membrane and organellemembranes


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Transport Functions of Cellular Membranes

A. Basic mechanisms to cross membranes

1. Passive, Active transport2.Role of integral proteins

3. Carriers, channels, facilitated diffusion

B. Active transport

1. Ion and molecular transport ATPases

a. P-class, F-class, V-class, ABC superfamily2. Gated channels

a. voltage, ligand and mechanical, nucleotide-gated channels.

C. Liposomes

1. Use in cell biology, therapeutically, cosmetically

D. Aquaporins

E. Gap junctions and plasmodesmata


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Thought questions:

How do molecules cross membranes?

Does it require energy to cross membranes?

Is it easier to cross the membrane of a

liposome or a plasma membrane?

Why?

Whats the biggest molecule that can crossa cell membrane?


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Transport processes

in a composite

eukaryotic cell.

Note: The ER, Golgi

and other vesiculuar

structures are not

shown.

Ions and many types of

molecules are

continually on the

move and crossing

membranes.

Aquaporins


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Transport functions of membranes.

Secretion

SecretionImport

Import


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?

How can water

cross a

lipid bilayer?


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Antiporter

[Facilitated]


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Saturation

Why does protein-mediated transport [facilitated diffusion] show saturation kinetics

while simple diffusion does not?


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Sodium, Magnesium , Chlorine and Calcium tend to enter.

Potassium has a tendency to exit . Energy is required to maintain these gradients.

This includes intracellular

compartments. i.e. ER


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Three ways of driving active transport.

Why is transport in a coupled carrier thermodynamically favorable?


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[carriers]

There are three types of transporter [carrier]-mediated transport:

Uniporter, Symporter and Antiporter. All are integral membrane proteins.

What is the thermodynamic reason for coupled- [symporter or antiporter]-transport?

Down an

Electrical gradient


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Carriers


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Sodium-driven glucose import and export: A symporter system.

Why does this type of transport work? Where is the energy for transport?


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Glucose or other transporters can be studied in liposomes.

Are these very good models? Why or why not?


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Channels

What are channels?

What is the difference

between channels and

carriers?


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Gated Channels


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Cyclic nucleotide gated ion channels [CNG channels]

What cellular functions are represented by these channels?

A. Cukkemane,R. Seifert & U. Kaupp. Trends in biochemical sciences 36:55-64. Jan. 2011


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How is the ph of the various compartmentsin a cell maintained?


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pH of different sub-cellular compartments in a typical mammalian cell.

The pH of the mitochondria refers to the matrix space contained within the inner

membrane.

How are these various pHs maintained?

J. Casey,S. Grinstein & J. Orlowski. Nature Reviews Molecular Cell Biology. 11:50-61. Jan. 2010


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Ion carriers that regulate cytoplasmic pH. The cytoplasm tends to acidify due to the activities of

various metabolic pathways. Several pH-regulatory transporters rectify this:

NHE: Na/H- exchangers. NBC: Na-/HCO3- exchangers. NDCBE: Na- dependent Cl-/HCO3

- exchangers.

AE: anion exchangers. PMCA: Plasma membrane Calcium-ATPases. NKA; Sodium/Potassium

ATPase

pumps. MCT:Monocarboxylate/H+ co-transporters.


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Control of Cellular Calcium. Note that Calcium channels

can be voltage and ligand- gated andcontrolled by external signals.


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Transient Receptor Potential Cation Channels. [TRPM]

The TRPM subfamily of cation channels are a subgroup

of the Transient-Receptor-Related [TRP] channels

found in almost all eukaryotic cells.

Why are they important?


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TRPM channels

These channels

can be gated

by calcium,

or by ADP-ribose,

NAD+,H2O2

or are gated

by internal or

external signals.

A. Fleig & R. Penner. Trends in Pharacological Sciences 25, 633- Dec. 2004


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TRPM2 channels can be gated by ADP-ribose [ADPR] ,

NAD+ or H2O2.

What does this signify with respect to its function?

See future lecture, but you dont have to wait. Look it up.

A. Fleig & R. Penner. Trends in Pharmacological Sciences 25, 633- Dec. 2004


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A. Fleig & R. Penner. Trends in Pharmacological Sciences. 25. 633-. Dec. 2004


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Inherited Disorders caused by Mutations of Ion Channels


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ATP-powered ion and small molecule pumps.


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Major classes of ATP-powered ion and small molecule pumps.


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Lehninger. Principles of Biochemistry. Third edition.

ABC transporters


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Cell Biology. Pollard & Earnshaw. Second Edition.2006.Saunders

Note that there are at least nine members of the ABC transporters

family.


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Structure of the V-ATPase.The complex is composed of a peripheral domain [V1] which is involved in ATP

hydrolysis and an integral domain [Vo] which is involved in protein transport across

the membrane.

The Vo domain includes a ring of proteolipids [c,c.c] that are adjacent to subunits

a and e. The V1and Vodomains are connected by a central stalk composed of

proteins D and F of V1and d of Vo.M. Forgac. Nature Reviews Molecular Cell Biology 8,917-929. Nov. 2007


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Regulation of V-ATPases

Depending on the cell type, V-ATPases can be regulated by:

a] Dissociation of the V1 and Vo domains mediated by glucose

depletion or interaction with aldolase.

b] Control by insertion into cell membranes. In the case ofepididymal clear cells the co-transport of Na+ and HCO3-

activate the second messenger cAMP which promotes the

endocytosis of the V-ATPase-containing vesicles

M. Forgac. Nature Reviews Molecular Cell Biology 8, 519-529. Nov. 2007


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The following nine slides deal

with the P-Type ATPase.


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P-type ATPases

Why are they important?

Why should cell biologists care

about them?


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General action of P-type ATPases


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Na+/K+ ATPase


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Whats

this?

Regulation of the NaK-ATPase by Cardiotonic Steroids,CTS


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High concentration of CTS

Low concentration of

CTS

g y

Little inhibition

J. Lingrel. Annu .Rev. Physiol. 72:395-412. 2010

High concentration of CTS:

Na+increases,Ca2+ increase

= muscle contraction.

Signaling pathways

activated.

Physiological levels of CTS.

Normal functions.


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Cardiotonic steroids


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ABC Transporters

ABC transporters can serve multiple roles.

1.Transport a variety of molecules

2.Flippase.

3.Secretion of a particular neurotransmitter.


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ATP-driven Chloride Channel.

What disease is caused by mutations of this channel?

The CFTR transporter for chloride is an ABC transporter.


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ABC transporter can act as a flippase.


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Families and domains of ABC Transporters Part 1.

Annual Review of Plant Biology 58: 347-375. 2007


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Some ABC transporters are called Multi-drug Resistance

transporters.

They are the cause of the secretion of drugs used to

kill toxic or cancerous cells.

Why would cells pump out beneficial drugs?

How would one get around this?


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Circumventing multi-drug resistance to anti-fungal toxins. A.The presence

of a drug e.g. azole drug designed to kill toxic fungal cells by ergosterolinhibition induces the transcription of the gene for an ABC transporter [PDR drug efflux

pump] which pumps out the azole drug. The cell is not killed.

B. Using a mult i funct ional drug [1.ergosterol inhibition, 2.production of toxic sterols,

3. induction of oxidative damage] effectively blocks the ABC transporter and

kills the cell.B. Monk and A. Goffeau. Science 321, 367-369. July 18, 2008


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Do cells secrete ATP?

Under what circumstances would cellssecrete ATP?

Is ATP a neurotransmitter?

Is ATP a signaling molecule?

If cells secrete ATP what transport

mechanism is used?

Vesicular? Pump?


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Purinergic signaling in plants and animals. ATP or ADP or other nucleotides including

cyclic nucleotides [cAMP,cGMP,etc.] can act as signaling molecules. ATP and others

must be secreted through channels, by exocytosis or through wounds.

K. Tanaka, et al. Trends in Cell Biology 20, No. 10. 601-608. Oct. 2010


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Secretion of ATP due to signals. Signaling by ATP or other nucleotides.

K.Tanaka, et al Trends in Cell Biology 20, 601-608,Oct. 2010 G. Burnstock. Physiol.Rev. 87. 2007


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Presynaptic nerve terminal - postsynaptic nerve cell.

This is just to remind us what a synaptic transmission is.


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ATP release by sub-epithelial sensory cells. Distension of viscera

stimulates the secretion of ATP. ATP acts as a neurotransmitter.Secreted ATP stimulates the receptors P2X3 or P2X2/3. This sends

a signal to the central nervous system. ATP is secreted by an ABC

transporter.

P.Bodin & G. Burnstock. Neurochemical Research 26, 959-969. Sept. 2001

For example shear force.

ATP ti i i


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ATP secretion is seen in

animals and plants.

Secreted ATP is recognized

by purinergic receptors.

ATP can be secreted using

ABC transporters.

What are possible uses for

this ATP?

ATP secretion in plant roots. Here it is visualized at the

tips by using the luciferin/luciferase detection method.

Why would plant roots secrete ATP?

S-Y Kim,M. Sivaguru,& G.Stacey. Plant Physiology 142, 984-992,

Nov. 2006

P. Bodin & G. Burnstock. Neurochemical

Research 26, 959-969. Sept. 2001.


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The transport of water


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Water is 55,000 mM in biological systems


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Aquaporins.

Note the six

transmembranehelices.

The smaller helices

HE and HB are

essentially

hydrophilic.

M. Sansom & R. Law. Current Biology 11, R71-R73, 2001


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Aquaporins

The hydrophilic helices

bend back in the cavity allowing

the passage of water.

T. Zeuthen. TIBS 26,No.2, Feb. 2001

Aquaporins in the human eye


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L.King,D. Kozono & Peter Agre*. Nature Reviews Molecular Cell Biology 5,687-698. Sept. 2004

* Nobel Prize for Aquaporins.


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Aquaporins in the human respiratory tract


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A. Verkman. Journal of Cell Science 124, 2107-2112. 2011


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Mammalian aquaporins


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What would be the result if the mRNA for an Aquaporin were injected

into these oocytes?


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The mRNA for Aquaporin was injected into Xenopus oocytes. The oocytesin the upper parts of each panel were placed in a hypotonic salt

solution. [0.035M]

Xenopus usually lives in an isotonic salt environment . [0.1 mM]

Why do the oocytes swell?

?

Role of V-ATPase in autophagy


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Amino acid efflux from the lysosome. What is required to pump the amino acids out

into the cytoplasm? What would be the consequences of a mutation in this pump?


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*

* Gap junctions transport molecules.

Gap junctions can transport molecules as large as 1.2nm in diameter,

or molecules from between 1200 and 2000 Daltons


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Transport in cells: Gap Junctions, Nuclear Pores and

Plasmodesmata.

How is transport regulated through these systems?


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Animal and Plant Junctions


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Junctions of epithelial tissues.


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Plasmodesmata of plant cells.

What is unique about this junction?


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Plasmodesmata Transport. Note that the endoplasmic

reticula [ER] of adjacent cells are connected.

Note that material can pass through the central cavity andthe ER

lumens.

What are the possible consequences in the event that one cell

undergoes endocytosis?


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Plasmodesmata: Mechanisms of cell-cell trafficking.

NCAPS are molecules that regulate vesicular traffic through plasmodesmata.

Microfilaments but not microtubules regulate vesicular traffic.


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Model of endo-exocytosis through plasmodesmata

A.B.

C.D.

E.

F.

G.

A ER B G l i C E t ti i l D A ti fil t E C ll ll F d t i G l b

H.

GG

transport functions slide show 2014

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