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.

Transport Functions/Properties of Membranes

Transport across the plasma membrane and organelle membranes

Transport Functions of Cellular Membranes A. Basic mechanisms to cross membranes 1. Passive, Active transport 2.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 superfamily 2. 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

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 cross a cell membrane?

Transport processes in a composite eukaryotic cell.

Note: The ER, Golgi and other vesiculuar structures are not shown.
Aquaporins

Ions and many types of molecules are continually on the move and crossing membranes.

Import
Import

Secretion Secretion

Transport functions of membranes.

?
How can water cross a lipid bilayer?

[Facilitated]

Antiporter

Saturation

Why does protein-mediated transport [facilitated diffusion] show saturation kinetics while simple diffusion does not?

This includes intracellular compartments. i.e. ER

Sodium, Magnesium , Chlorine and Calcium tend to enter. Potassium has a tendency to exit . Energy is required to maintain these gradients.

Three ways of driving active transport. Why is transport in a coupled carrier thermodynamically favorable?

Down an Electrical gradient

[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?

Carriers

Sodium-driven glucose import and export: A symporter system. Why does this type of transport work? Where is the energy for transport?

GLUT1 and GLUT2 are members of a family of glucose uniporters. GLUT1 is in erythrocytes; GLUT2 is in liver cells. Why are their rates of glucose uptake different? Erythrocytes have no nuclei or other organelles, so how is glucose uptake regulated?

Glucose or other transporters can be studied in liposomes. Are these very good models? Why or why not?

Channels What are channels?

What is the difference between channels and carriers?

Gated Channels

Stereocilia are stimulated by mechanically-gated channels

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

How is the ph of the various compartments in a cell maintained?

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

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.

Control of Cellular Calcium. Note that Calcium channels can be voltage and ligand- gated and controlled by external signals.

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?

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

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

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

Inherited Disorders caused by Mutations of Ion Channels

ATP-powered ion and small molecule pumps.

F-ATPases can make ATP

V-ATPases require ATP

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

ABC transporters

Lehninger. Principles of Biochemistry. Third edition.

Note that there are at least nine members of the ABC transporters family.
Cell Biology. Pollard & Earnshaw. Second Edition.2006.Saunders

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 V1 and Vo domains are connected by a central stalk composed of proteins D and F of V1 and d of Vo.
M. Forgac. Nature Reviews Molecular Cell Biology 8,917-929. Nov. 2007

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 of epididymal clear cells the co-transport of Na+ and HCO3activate 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

Protein C

Regulation of V-ATPase activity. Dissociation method. The V-ATPase is composed of two multi-protein subunits, Vo and V1 . The V-ATPase can undergo reversible dissociation into a V-complex that lacks protein C. Vo remains in the membrane; protein C dissociates from V1. V1 C dissociates from Vo. This can happen during glucose depletion or by altered interaction with aldolase. The dissociation requires intact microtubules. The complex RAVE [Skp1,Rav1,Rav2] regulates the disassociation and binds to the released subunit. [V1{-C}]. The RAVE complex also regulates the assembly of the V-ATPase. In some cell types assembly requires the activity of phosphoinositide 3-kinase. [PI3K]
M. Forgac. Nature Reviews Molecular Cell Biology. 8, 917-929. Nov. 2007

The following nine slides deal with the P-Type ATPase.

P-type ATPases Why are they important? Why should cell biologists care about them?

General action of P-type ATPases

Na+/K+ ATPase

Whats this?

Regulation of the NaK-ATPase by Cardiotonic Steroids,CTS

High concentration of CTS

High concentration of CTS: Na+ increases,Ca2+ increase = muscle contraction.

Signaling pathways activated.

Physiological levels of CTS. Normal functions.

Low concentration of CTS

Little inhibition

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

Cardiotonic steroids

ABC Transporters

ABC transporters can serve multiple roles. 1.Transport a variety of molecules 2.Flippase.

3.Secretion of a particular neurotransmitter.

ABC transporters move many types of molecules including ATP. What effect do these transporters have on chemotherapy?

ATP-driven Chloride Channel. What disease is caused by mutations of this channel?

The CFTR transporter for chloride is an ABC transporter.

ABC transporter can act as a flippase.

Families and domains of ABC Transporters Part 1.

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

Families of ABC Transporters Part 2.

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

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?

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 ergosterol inhibition 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 multifunctional 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

Do cells secrete ATP?

Under what circumstances would cells secrete ATP? Is ATP a neurotransmitter?

Is ATP a signaling molecule?

If cells secrete ATP what transport mechanism is used? Vesicular? Pump?

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

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

Presynaptic nerve terminal - postsynaptic nerve cell.

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

Secreted ATP in animal and plant cells In animal cells vascular shear stress In plant cells ATP is and hypoxia can stimulate the uptake secreted into the extracellular matrix of ATP secreted from platelets and due to wounding, during osmotic endothelial cells for vasoconstriction stress, through exocytosis or vasodilatation . during cell wall synthesis or mechanical stimulation. [touch]

G. Burnstock. Nature Medicine 12,16-17. Jan. 2006

S. Roux & I. Steinebrunner. Trends Plant Sci.12,522-527. Nov. 2007

For example shear force.

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

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?

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

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

The transport of water

Water is 55,000 mM in biological systems

Aquaporins. Note the six transmembrane helices. The smaller helices HE and HB are essentially hydrophilic.

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

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

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

* Nobel Prize for Aquaporins.

Aquaporins in the human respiratory tract

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

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

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

Mammalian aquaporins

What would be the result if the mRNA for an Aquaporin were injected into these oocytes?

The mRNA for Aquaporin was injected into Xenopus oocytes. The oocytes in 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

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?

* Gap junctions transport molecules. Gap junctions can transport molecules as large as 1.2nm in diameter, or molecules from between 1200 and 2000 Daltons

Transport in cells: Gap Junctions, Nuclear Pores and Plasmodesmata. How is transport regulated through these systems?

Animal and Plant Junctions

Junctions of epithelial tissues.

Gap junction

Plasmodesmata of plant cells. What is unique about this junction?

Plasmodesmata Transport. Note that the endoplasmic reticula [ER] of adjacent cells are connected.
Note that material can pass through the central cavity and the ER lumens. What are the possible consequences in the event that one cell undergoes endocytosis?

Plasmodesmata: Mechanisms of cell-cell trafficking. NCAPS are molecules that regulate vesicular traffic through plasmodesmata. Microfilaments but not microtubules regulate vesicular traffic.

G G
A. B.

C.
D.

E.
F.

H.

G.

Model of endo-exocytosis through plasmodesmata

A:ER B: Golgi H: desmotubule C: Exocytotic vesicle D: Actin filament E: Cell wall F: endocytosis G: plasma membrane