Transport Across Membranes

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Objectives Be familiar with mechanisms of transporting material across the

membrane and key examples Know the differences between passive and active transport Osmosis

Know terms hypertonic, hypotonic, and isotonic Diffusion across membranes

Effect of size, polarity and charge Facilitated diffusion (channels and carriers)

Porins & Aquaporins Uniporter (Examples: Glut permeases) Co-transporters

Antiporter (Example: Anion exchange protein) Symporter (Example: Sodium- glucose symporter )

Channels Ion channels

Ligand-Gated, Mechanosensitive & Voltage-Gated

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Objectives Indirect and direct active transport

Example of direct active transport: Na+/K+ Pump Example of indirect active transport: Na+/glucose symporter

Know how glucose is transported from the lumen of the intestine, through intestinal epithelial and into the extracellular fluid on the basal-lateral side of the epithelium

Know the different types of endocytosis Phagocytosis, clathrin-dependent and –independent pinocytosis Know Familial Hypercholesterolemia Know the three fates of receptors after endocytosis

Clinical Correlations: Cystic Fibrosis

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Cells and Transport processes

Membranes are selectively permeable and control the passage of specific molecules and ions from one side of the membrane to the other

Cells need a higher concentration of solutes within the cell than found typically outside the cell in order for many reactions to occur at reasonable rates

Solutes cross membranes by simple diffusion, facilitated diffusion, and active transport

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Table 11-1 Molecular Biology of the Cell (© Garland Science 2008)5

Relative permeability of lipid bilayers to different classes of molecules

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Figure 11-4a Molecular Biology of the Cell (© Garland Science 2008)

How molecules move across the membrane

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Simple Diffusion: Unassisted movement down the gradient

Diffusion always moves solutes toward equilibrium

Diffusion always proceeds from regions of higher to lower free energy Equilibrium is the lowest

energy state Osmosis is the diffusion of

water across a differentially permeable membrane

Simple diffusion is limited to small, nonpolar molecules

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Simple diffusion is limited to small, nonpolar molecules

Diffusion is always movement toward equilibrium (minimum free energy)

Solute size (cut-off size is approx. 200 amu) Generally, membranes are more permeable to smaller

molecules than larger ones For example: Glucose is too large

Solute polarity Permeable to nonpolar, more impermeable to polar

Ion Permeability Membranes are impermeable to ions

Small nonpolar molecules: oxygen, carbon dioxide and ethanol can easily diffuse across membrane

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Osmosis

Water diffuses from where the solute concentration is lower, across a differentially-permeable membrane, to where the solute concentration is higher.

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Osmosis (cont)

Osmolarity: solute concentration on one side of a membrane relative to that on the other side of the membrane; drives the osmotic movement of water across the membrane

Hypertonic: a solution with a higher solute concentration than inside the cell

Isotonic: a solution with an equal solute concentration as that inside the cell

Hypotonic: a solution with a lower solute concentration than inside the cell

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Osmosis

There are typically more solutes inside than outside of a cell

Plant cells and bacteria have cell walls Animal cells have ion pumps

Figure 11-16 Molecular Biology of the Cell (© Garland Science 2008)

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Facilitated Diffusion: Protein-mediated movement down the

gradient Facilitated diffusion does not require input of

energy

Two main classes of proteins involved with facilitated diffusion: Carriers and channels

Carrier proteins and channel proteins facilitate transport by different mechanisms Carrier proteins (transporters or permeases) bind

one or more solute molecules on one side of the membrane and undergo conformational change to deliver solute to the other side of membrane

Channel proteins: form hydrophilic channels, often transport ions

Ion channels Porins Aquaporins 13

Carrier Proteins/Permeases/Transporters

Carrier proteins probably alternate between two conformational states

Carrier proteins are analogous to enzymes in their specificity and kinetics Some carriers are extremely specific

Kinetics of carrier protein function Display saturation kinetics and can be

subjected to competitive inhibition Carrier proteins transport either one or two

solutes Uniport: single solute Cotransport: two solutes (couple)

Symport: both in the same direction Antiport: solutes are transported in opposite directions

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Figure 11-6 Molecular Biology of the Cell (© Garland Science 2008)

Transporters Display Saturation Kinetics

Note the plateauThis indicates saturation

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The Glucose Transporter FamilyGLUT-1 - GLUT-5

The initial event in the cellular metabolism of glucose is its transport across the cell membrane into the cytoplasm of the cell.

This step is performed by the GLUT permeases Members of the family include:

GLUT-1: a uniport carrier found in red blood cells and neurons where it functions in the basal transport of glucose into the cell

Glucose levels higher in the blood than inside the cells Low cellular glucose is maintained by hexokinase which

phosphorylates glucose to glucose-6-phosphate (traps glucose in cell this way, cell lacks transporter for phosphorylated sugars)

GLUT-2: a uniport carrier found in hepatocytes, where it functions in bidirectional glucose transport

Also found in other cells such as kidney cells, surface absorptive cells and pancreatic beta cells

GLUT-3: a uniport carrier found in neurons GLUT-4: a uniport carrier found in adipocytes, skeletal muscle

cells, and cardiac myocytes where it functions in the insulin-stimulation transport of glucose into the cell

GLUT-5: a uniport carrier found on surface absorptive cells of the small intestine mucosa where it functions in the transport of fructose into the cell

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The erythrocyte anion exchange protein:

An antiport carrier The erythrocyte anion exchange protein is also called band 3

protein, and chloride-bicarbonate exchanger

Solute binding site of the anion exchange protein interacts with different ions on opposite sides of the membrane

Necessary to prevent net charge imbalance (one negative ion in for one negative ion out)

The anion exchanger transport Cl- across the membrane by countertransport with HCO3

- (called the chloride shift) Important in the transport of carbon dioxide in the body and for

helping to regulate pH

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The erythrocyte plasma membrane provides examples of transport

mechanisms

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Direction of oxygen, carbon dioxide, and bicarbonate transport in

erythrocytes In the lung, oxygen diffuses from the

inhaled air to the cytoplasm of the cell In the capillaries, oxygen is release by

hemoglobin and diffuses from the cytoplasm into the blood plasma

Carbon dioxide is often transported in the form of bicarbonate ion

Carbon dioxide converted to bicarbonate in the capillaries and released into blood plasma

In the lungs, bicarbonate is imported into the cytoplasm and converted to carbon dioxide

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Transporters in plasma membrane regulate cytosolic

pH Most cells have one or more Na+ -driven

antiporters in their plasma membrane that help to maintain the cytosolic pH at ~7.2

These transporters use energy stored in the Na+ gradient to pump out excess H+.

Two mechanism H+ directly transported out by Na+ - H+

exchanger Neutralize H+ in the cytosol with HCO3

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Na+– driven Cl--HCO3-- exchanger

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Channel proteins facilitate diffusion by forming hydrophilic transmembrane

channelsThree kinds of channel proteins: Ion channels: Transmembrane proteins that allow rapid

passage of specific ions

Voltage-gated Ligand-gated Mechanosensitive

Porins: Transmembrane proteins that allow rapid passage of various solutes

Beta barrel transmembrane region creates water-filled pore at its center

Aquaporins: Transmembrane channels that allow rapid passage of water

Can facilitate transport at a rate of several billion water molecules per second

Found in certain tissues such as the proximal tubules of the kidneys that reabsorb water as part of urine formation

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Ion Channel Proteins Ion channel proteins form hydrophilic pores to transport

inorganic ions across the membrane They only participate in passive transport

(facilitated diffusion) whereby molecules are transported “downhill” of the concentration and membrane potential; i.e. electrochemical gradient

Ion channels are selective and gated (open briefly then close)

Stimuli that open gates include: Mechanical stress (mechanical-gated ion channels) Changes in voltage across the cell membrane (voltage-gated

ion channels) Ligand binding (ligand-gated ion channels)

One of the most important types of ligand-gated ion channels is the transmitter-gated ion channels that bind neurotransmitters and mediate ion movement

Depending on the ion involved, it may have an excitatory or inhibitory effect25

Transmitted-gated ion channels

(Ligand-gated) Nicotinic acetylcholine (nACh) receptor Glutamate receptors

N-methyl-D-aspartate (NMDA) receptor Kainate receptor a-amino-3-hydroxy-5-methyl-4-isoxazole propionic

acid (AMPA) receptor 5-Hydroxytryptamine3 (5-HT3) serotonin

receptor Purinergic2x receptor Gamma-aminobutyric acidA (GABAA) receptors Glycine receptor

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Clinical CorrelationAn 11-year-old white female is brought to the emergency room by her parents because of fever, difficulty breathing, and a productive cough with greenish sputum. Her parents are of northern European descent. She has a history of recurrent lower respiratory tract infections and foul-smelling diarrhea since infancy. Physical examination finds tachycardia, mild cyanosis; malnourishment; clubbing of fingernails; nasal polyps; hyperresonance to lung percussion with barrel-shaped chest. Laboratory results reveal high sweat sodium and chloride concentrations in sweat test; sputum culture is positive for Pseudomonas aeruginosa, Haemophilus influenzae and Staphylococcus aureus.1) What is the most likely diagnosis for this patient?

2) What is the molecular pathogenesis of this disorder?27

Cystic Fibrosis

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Clinical Connection - Answers The patient has Cystic Fibrosis

Caused by an autosomal recessive mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene

An ABC transporter family membre Most common CFTR mutation in Caucasians is DF508

Mechanism: The mutation results in inability of cells to transport

chloride and water to body secretions Results in increased frequency of respiratory

infections Also causes pancreatic insufficiency Males often are sterile End stage, progressive lung disease is the principle

cause of death http://cpmcnet.columbia.edu/dept/cs/thoracic/cf.html29

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Image from: http://en.wikipedia.org/wiki/File:Cystic_fibrosis_manifestations.png

Why is the skin salty?

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Image from:http://jillclare.tripod.com/cf/

Active transport: Protein-mediated movement up the gradient

Active transport requires expenditure of energy! Unlike simple and facilitated diffusion (both

nondirectional), active transport has directionality usually a unidirectional process

Three major functions of Active Transport:1) Uptake of essential nutrients2) Removal of secretory products and waste3) Maintain nonequilibrium intracellular concentrations of

ions The coupling of active transport to an energy source may

be direct or indirect Direct active transport depends on four types of

transport ATPases Indirect active transport is driven by ion gradients

2/3 of body’s energy consumed to maintain gradients of ions such as H+, K+, Na+ and Ca+

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Direct versus Indirect

Direct active transport: the accumulation of solute molecules or ions on one side of the membrane is coupled directly to an exergonic chemical reaction (ATP hydrolysis)

Indirect active transport: depends on the cotransport of two solutes, with the movement of one solute down its gradient driving the movement of the other solute up its gradient (usually sodium or proton ions) Animal cells usually depend on Sodium ion (Na+)

gradients as the driving force for indirect active transport

Plant, bacteria and fungi usually depend on proton (H+) gradients

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Examples of active transport

Direct Active Transport:

Na+/K+ PumpIndirect Active

Transport: Na+/glucose

symporter

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Direct Active Transport: Na/K Pump

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Na+-K+ ATPase: an antiporter carrier protein found in almost all cells that pumps Na+ out of the cell and K+ into the cell to maintain a low intracellular [Na+].

P-type pumpOubain is a specific inhibitor that competes for K+-binding site. Cardiac glycosides (digoxin and digitoxin) are Na+-K+ ATPase blockers that elevate intracellular Na+ levels within cardiac myocytes. The elevated Na+ overwhelms the Na+- Ca2+ Exchanger so that more Ca2+ can be reaccumulated by the sarcoplasmic reticulum. During the next contraction, more Ca2+ is released from the sarcoplasmic reticulum, which increased the force of contraction. Cardiac glycosides are used in congestive heart failure to increase the force of contraction

EFFECT OF DIGOXIN

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Indirect Transport: Na/glucose symporter

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Na+-glucose cotransporter: a symporter carrier protein most commonly found in surface absorptive cells of the small intestine mucosa and the simple columnar epithelium of the proximal convoluted tubule of the kidney that transport Na+ across the luminal membrane into the cytoplasm by cotransport with glucose.

Transport of large molecules:

Endocytosis – Uptake of macromolecules from the extracelluar surroundings by localized regions of plasma membrane Phagocytosis – endocytosis of large particulate

substances Pinocytosis – endocytosis of fluid and dissolved

solutes Receptor-mediated – binding of ligands to

receptors triggers vesicle formation Exocytosis – secretion of macromolecules by

transport vesicles Endocytosis & exocytosis are mechanisms that

involve movement into and out of the lumen of the endomembrane system Not movement directly across membrane That is, substances enter the Endomembrane System but

not the Cytoplasm38

Endocytosis Endocytosis allows cells to take up

macromolecules, fluids, and large particles such as bacteria.

The material is surrounded by an area of plasma membrane, which buds off inside the cell to form a vesicle containing the ingested material.

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Phagocytosis Phagocytosis (cell eating) occurs in specialized cell

types. Binding of the particle to receptors on the cell surface

triggers the extension of pseudopodia, which surround the particle and fuse to form a large vesicle called a phagosome.

Phagosomes fuse with lysosomes to form phagolysosomes, in which the material is digested by acid hydrolases

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Phagocytosis

Many amoebas use phagocytosis to capture food particles such as bacteria.

In multicellular animals, phagocytosis is used as a defense against invading microorganisms, and to eliminate aged or damaged cells

In mammals, macrophages and neutrophils (white blood cells) are the “professional phagocytes.”

They remove microorganisms from infected tissues, and macrophages eliminate aged or dead cells from tissues throughout the body.

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Receptor-mediated endocytosis

Receptor-mediated endocytosis is a mechanism for selective uptake of specific macromolecules.

Macromolecules bind to cell surface receptors concentrated in specialized regions called clathrin-coated pits.

The pits bud from the membrane with the help of dynamin, to form small clathrin-coated vesicles; these then fuse with early endosomes.

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After internalization, clathrin-coated vesicles shed their coats and fuse with early endosomes—vesicles with tubular extensions at the cell periphery.

The molecules are sorted, recycled to the plasma membrane, or remain in the early endosomes as they mature to late endosomes and lysosomes for degradation.

Early endosomes maintain an acidic internal pH (about 6.0 to 6.2) by the action of a membrane H+ pump.

This causes dissociation of many ligands from their receptors.

Receptors can be returned to the plasma membrane via transport vesicles. Ligands such as LDL remain and are degraded to release cholesterol.

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Recycling of the receptor molecules is important in replacing plasma membrane that is internalized during endocytosis.

As endosomes mature, lipid composition of the membrane changes in preparation for fusion with vesicles carrying lysosomal hydrolases from the trans Golgi.

Late endosomes are more acidic (pH 5.5 to 6.0). They mature into lysosomes (pH 5) where the endocytosed materials are degraded by acid hydrolases.

Some receptors are not recycled, but are degraded in lysosomes. Receptors for several growth factors are degraded in

this way, which eventually stops the the cell’s response to growth factors: receptor down-regulation.

Receptor-mediated endocytosis of LDL

Receptor-mediated endocytosis was first elucidated in studies of patients with familial hypercholesterolemia.

Cholesterol is transported through the bloodstream mostly in the form of low-density lipoprotein, or LDL.

LDL binding sites on normal cells were determined by adding radiolabeled LDL to cell cultures by Brown and Goldstein in 1974.

Cells of FH patients did not bind LDL.

They then established that the LDL receptor is internalized by endocytosis from coated pits.

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Clinical Correlation: Familial Hypercholesterolemia

Hypercholesterolemia causes the formation of artherosclerotic plaques and arthersclerosis (hardening of the arteries).

Familial hypercholesterolemia: genetic predisposition to high blood cholesterol levels and heart disease caused by an inherited defect in the gene encoding for the LDL receptor

Low-density lipoproteins are not taken up by the cells and this induces the cell to produce cholesterol.

LDL is composed of apoprotein B-100, ~1500 esterified cholesterol molecules (FA attached), free cholesterol and a phospholipid monolayer.

LDL receptor recognizes apoprotein B-100.

Cholesterol, LDL receptor and endocytosis

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Familial hypercholesterolemia

• Familial hypercholesterolemia (FHC) is a hereditary (I = 1/500) hyperlipidemic condition, characterized by an elevated level of plasma cholesterol

- FHC patients are at increased risk of developing atherosclerosis, often leading to myocardial infarction ~ 5% of patients with MI over the age of 60 years have FHC

- Plasma cholesterol level in heterozygotes - Total cholesterol 300 – 600 mg/dL

- LDL cholesterol > 160 mg/dL- Cholesterol levels in homozygous patients - Total cholesterol 600 – 1200 mg/dL

- Homozygotes are at much greater risk of developing widespread atherosclerosis

- Xanthomas more prominent in Homozygotes- Ischemic heart disease often develop in

homozygotes before the age of 20- Often leading to death at early age

Type II Hyperlipoproteinemia Xanthelasma

Cholesterol accumulation in soft tissues and skin, producing xanthomas is often seen in Familial Hypercholesterolemia

Cutaneous xanthomas in a familial hypercholesterolemia homozygote. B Cutaneous xanthomas in homozygous familial hypercholesterolemia

The receptor-mediated endocytosis of LDL. Note that LDL dissociates from its receptors in the acidic environment of the early endosome. After a number of steps, the LDL ends up in the lysosomes, where it is degraded to release free cholesterol. In contrast, the LDL receptors are returned to the plasma membrane via clathrin-coated transport vesicles that bud off from the tubular region of the early endosomes. Whether it is occupied or not, an LDL receptor typically makes one round trip into the cell and back to the plasma memebrane every 10 minutes, making a total of several hundred trips in its 20-hour lifespan.

Figure 13-53 Molecular Biology of the Cell (© Garland Science 2008)

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The cell biology and biochemical role of the LDL receptor and the six classes of mutations that alter its function. After synthesis in the endoplasmic reticulum (ER), the receptor is transported to the Golgi apparatus and subsequently to the cell surface. Normal receptors are localized to clathrin-coated pits, which invaginate, creating coated vesicles and then endosomes, the precursors of lysosomes. Normally, intracellular accumulation of free cholesterol is prevented because the increase in free cholesterol (A) decreases the formation of LDL receptors, (B) reduces de novo cholesterol synthesis, and (C) increases the storage of cholesteryl esters. The biochemical phenotype of each class of mutant is discussed in the text. ACAT, acyl coenzyme A:cholesterol acyltransferase; HMG CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase.

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LDL Receptor Mutations

Clathrin-independent endocytosisvia caveola Caveolae are small invaginations

of the plasma membrane organized by caveolin.

Caveolins are a family of proteins that interact with cholesterol in lipid rafts and with each other

Caveolae carry out receptor-mediated endocytosis via specific transmembrane receptors.

The caveolae lipids and caveolin itself also serve as “receptors” for uptake of specific molecules, including high density lipoprotein (HDL).

Other endocytosis pathways are independent of both clathrin and calveolin.

Additionally, large vesicles can mediate uptake of fluids in a process known as macropinocytosis.

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54Figure 13-52 Molecular Biology of the Cell (© Garland Science 2008)

Three possible fates for transmembrane receptor proteins

after endocytosis1. Retrieved receptors are

returned to the same plasma membrane domain from which they came (recycling)

2. Receptors are sent to a different domain of the plasma membrane (transcytosis)

3. Receptors that are not specifically retrieved from the endosomes follow the pathway from the endosomal compartment to lysosomes, where they are degraded.

Transcytosis In polarized epithelial

cells, receptors can be transferred across the cell to the opposite domain of the plasma membrane—transcytosis.

In some cells, this is an important membrane protein sorting mechanism; and also used to transfer extracellular macromolecules across epithelial cell sheets.

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56Figure 13-60 Molecular Biology of the Cell (© Garland Science 2008)

Transcytosis transfers macromolecules across epithelial

cell sheets Recycling endosomes form a way-station on the transcytosis pathway.

In the example shown here, an antibody receptor on a gut epithelial cell binds antibody and is endocytosed, eventually carrying the antibody to the basolateral plasma membrane

Recycling of Synaptic Vesicles

In nerve cells, after synaptic vesicles release their neurotransmitters, they are recovered in clathrin-coated vesicles which fuse with early endosomes.

The synaptic vesicles are regenerated by budding from endosomes

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