Transmembrane Transport of Ions and Small Molecules

MOLECULAR CELL BIOLOGY SIXTH EDITION

MOLECULAR CELL BIOLOGY SIXTH EDITION

Copyright 2008 ©W. H. Freeman and Company

CHAPTER 11

Transmembrane Transport of Ions and Small Molecules

CHAPTER 11

Transmembrane Transport of Ions and Small Molecules

Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira

© 2008 W. H. Freeman and Company

A study of mutant zebrafish with pale stripes led to the identification of a sodium/calcium transporter that regulates the

darkness of human skin

The phospholipid bilayer is a barrier that controls the transport of molecules in and out of the cell.

Gases diffuse freely, no proteins required.

Water diffuses fast enough that

proteins aren’t required for transport

Sugars diffuse very slowly so proteins are involved in transport.

Charged molecules are virtually impermeable.

Studies of synthetic lipid bilayers help define which types of transport will require the activity of a protein. Hence, transport of an ion should require a protein.

Only small hydrophobic molecules cross membrane

The bilayer is permeable to:

Small hydrophobic molecules

Small uncharged polar molecules

The bilayer is impermeable to:

Ions

Large polar molecules

THEREFORE, need membrane proteins to transport most molecules and all ions across biomembranes

KEY CONCEPTS

Selective transport across the lipid membrane requires transport proteins

Transport proteins are integral membrane proteins that move molecules and ions

There are two classes of transport proteins:

transporters (pumps) and channels

Most small molecules did not across membrane

Transporter protein

Also called

Na+/K+ ATPase sodium-amino

acid transporter

Three main class of membrane protein 1.ATP- power pump( carrier, permease)

couple with energy source for active transport binding of specific solute to transporter which

undergo conformation change 2. Channel protein (ion channel)

formation of hydrophilic pore allow passive movement of small inorganic molecule

3. Transporters uniport

symport antiport

Partition Coefficient

Permeability coefficients (in cm/sec) through synthetic lipid bilayers

Product of the concentration difference (in mol/cm3) and permeability coefficient (in cm/sec) gives the flow of solute in moles per second per square centimeter of membrane

油品分配係數

Cell membrane

•Barrier to the passage of most polar molecule

•Maintain concentration of solute Diffusion rate depends on :

1. Concentration gradient or electrochemical gradient 2. Hydrophobicity

i.e. higher partition coefficient 3. Particle size

Membrane proteins mediated transport of most molecules and all ions across biomembrane

The rate at which a molecule diffuses across a synthetic lipid bilayer depend on its size and solubility

The smaller the molecule and the less polar it is, the more rapidly it diffuses across the bilayer

Overview of membrane transport proteins

1. All transmembrane proteins

2. Some transport has ATP binding sites

3. Move molecules uphill (向上)

against its gradient

Differences

1. Transporters=

-uniporters transport a single molecule down its gradient (passive)

-co-transporters couple movement of a molecule down its gradient with moving a molecule up its gradient (active) 2. Pumps = hydrolyze ATP to move small molecules/ions up a

concentration gradient or electric potential (active)

3. Channels = transport water/ions/small molecules down their concentration gradients or electric potentials (passive)

The four mechanisms of small molecules and ions are transported cross cellular membranes

Ion → force

促進 主動

If transport substance carries a net charge, its movement is influenced by both its concentration gradient and the

membrane potential, the electric potential (voltage) across the membrane.

Substance concentration + electric potential =

electrochemical gradient. Determines the energetically favorable direction of transport a charged molecule across a membrane.

Copyright © 2009, Dr. Salme Taagepera PhD. All rights reserved.

Passive transport driven by:

Concentration gradient

(affects both uncharged and charged solutes)

Electrical gradient

(affects only charged solutes)

“Electrochemical gradient”

+

被動 Facilitated Diffusion

Passive transport: no metabolic energy is needed because the solute is moving down its concentration gradient.

•In the case of an uncharged solute, the concentration of the solute on each side of the membrane dictates the direction of passive transport.

Active transport: metabolic energy is used to transport a solute from the side of low concentration to the side of high concentration.

Two types of transport are defined by whether metabolic energy is expended to move a solute across the membrane.

Facilitated Diffusion

Free Diffusion

A. Non-channel mediated

– lipids, gasses (O2, CO2), water B. Channel mediated

– ions, charged molecules

Facilitated diffusion

Carrier mediated

– glucose, amino acids

[ECF]

Extracellular fluid

Facilitated Diffusion

Rate of diffusion is determined by:

1. concentration gradient 2. amount of carrier protein

3. rate of association/dissociation

Unique features for Uniport transport:

1. Higher diffusion rate for uniport than passive diffusion.

2. Transported molecules

never enter membrane and Irrelevant (無關) to the

partition coefficient. (did not cross membrane)

3. Transport rate reach V_max when each uniport

working at its maximal rate 4. Transport is specific. Each

uniport transports only a single species of

molecules or single or closely related molecules

Several feature distinguish uniport transport from passive diffusion

GLUT: glucose transport

Need a carrier protein

Uniport transport of glucose and water

Glucose: utilize glucose as a source for ATP production

Water: utilize aquaporins to increase the rate of water movement.

H2O: hybrophilic, did across membrane

Families of GLUT proteins(1-12)

Highly homologous in sequence, and contain 12 membrane-spanning α - helices.

Different isoforms → different cell type expression, and different function GLUT2: express in liver cell ( glucose storage)

and ß cell( glucose uptake) pancreas GLUT4: found in intracellular membrane,

increase expression by insulin for remove the glucose from blood to cell GLUT5: tansport fructose.

Other isoforms: ???

Glucose transporter (GLUT): Facilitated Diffusion

Mammalian glucose transporters

Name Tissue distribution Proposed function

Glut1 all fetal and adult tissues basal glucose transport Glut2 hepatocytes, pancreatic β-cells transepithelial transport

intestine, kidney from and to the blood Glut3 widely distributed basal glucose transport

mostly in brain

Glut4 skeletal muscle, heart, adipocytes insulin-dependent transport Glut5 intestine, lesser amounts few others fructose transport

Glut7 gluconeogenic tissues mediates flux across endoplasmic reticulum Glut8 preimplantation blastocyst embryonic insulin-

dependent transport

Oded Meyuhas

Each GLUT protein contains 12 membrane-spanning alpha helices Differentially expressed

– EXAMPLE: GLUT4 is only expressed in fat and muscle cells

• Fat and muscle cells respond to insulin by increasing their uptake of glucose, thereby removing glucose from the blood

• In absence of insulin = GLUT4 on intracellular membranes

In presence of insulin = GLUT4 found on cell surface

• QUESTION: Defects in directing GLUT4 to the cell surface can cause what common disease?

Type II diabetes, high blood glucose

Copyright © 2009, Dr. Salme Taagepera PhD. All rights reserved.

GLUT1 is responsible for transporting glucose across the blood- brain barrier → GLUT1 provides glucose for the brain

GLUT1 deficiency syndrome:

• Brain does not obtain enough glucose from the blood

• Symptoms: seizures, developmental delay, motor disorders

• Treatment: ketogenic diet (high fat/low carb diet)

GLUT1 deficiency syndrome

Glucose

Transporter proteins

(membrane protein) can be enriched within artificial membrane

Chloroform and methanol (3:1)

Phospholipid

spontaneously form bilayers

Liposome containing a single type of transport protein are very useful in studying

functional properties of transport proteins

• It is a major experimental tool to study the biochemistry of transport protein function in vitro

• Widely used as a drug

delivery system and for gene transfection

Movement of water

Osmosis: movement of water across semipermeable membrane

Osmotic pressure: hydrostatic pressure uses to stop the net flow of water

When C_B concentration > C_A

Osmotic pressure π=RT( C_B -C_A )

Hypertonic solution: the concentration is higher than cytosol Isotonic solution: equal to cytosol

Hypotonic solution: lower; and water move to cytosol

Animal cell ace a problem in maintaining their cell volume within a limited range, thereby avoiding lysis

Plant cell: has cell wall → prevent cell shape

Turgor pressure (膨壓): osmotic pressure, plasma membrane against water into the cytosol and then into the vacuole

turgor pressure supplies rigidity

The large forces of turgor pressure are resisted by the strength of cellulose microfibrils in the cell wall

Hypertonic external solution: concentration of water is low relative to its concentration inside the cell

Water moves out down its concentration gradient and the cell shrinks

Hypotonic external solution: concentration of water is high relative to its concentration inside the cell

Water moves in down its concentration gradient and the cell swells

When the Na⁺ – K⁺ pump stops, Na⁺ goes into the cell along its concentration gradient

This adds to the solute concentration in the cytosol Water moves into the cell along its concentration gradient and the cell bursts

Water draw is equal inside and outside

Water cross membrane is very lower

Water cross membrane via specific channel- aquaporins

Aquaporins increase the water permeability of cell membrane

Aquaporin 1 erythrocyte

Aquaporin2 kidney cells resorb water from urine; mutation → diabetes insipidus → large volume urine

Expression of aquaporin by frog oocytes increases their permeability

control

Injection aquaporin mRNA

Egg move to hypotonic environment

尿崩病

Water channel protein ( aquaporin)

Tetrameric protein

6 α-helices for each subunit

2-nm-long water selective gate 0.28nm gate width

Highly conserved arginine and

histidine in the gate

H₂ O for HO bonding

with cystein

Aquaporins

Aquaporins are membrane water channels that play critical are membrane water channels that play critical roles in controlling the

roles in controlling the waterwater contents of cells.contents of cells.

Water

Water crosses the crosses the hydrophobic membrane either by simple hydrophobic membrane either by simple diffusion or through a facilitative transport mechanism diffusion or through a facilitative transport mechanism mediated by these

mediated by these specialized proteinsspecialized proteins. . These protein channels are

These protein channels are widely distributedwidely distributed in all kingdoms of in all kingdoms of life, including bacteria, plants, and mammals.

life, including bacteria, plants, and mammals.

Important in

Important in osmotic regulationosmotic regulation, acting to prevent bursting of , acting to prevent bursting of the cells whenever there are changes of the exterior salt the cells whenever there are changes of the exterior salt

concentration.

concentration.

ATP powered pump

1. P- class

2α, 2β subunit; can phosphorylation

i.e. Na⁺-K⁺ ATP ase, Ca⁺ATP ase, H⁺pump 2. F-class

• locate on bacterial membrane , chloroplast and mitochondria

• pump proton from exoplasmic space to cytosolic for ATP synthesis

3. V-class

maintain low pH in plant vacuole similar to F-class

4. ABC (ATP-binding cassete) superfamily

several hundred different transport protein

Different classes of pumps exhibit characteristic structure and functional properties

Specific Ion binding site

Must phosphorylation

Transport process requires ATP hydrolysis in which the free energy is liberated by breakdown of ATP into ADP and

phosphate.

V-class H+ ATP ase pump protons across lysosomal and vacuolar membrane

inside

inside

ATP powered pump 1. P- class

2α, 2β subunit

i.e. Na

-K

ATP ase, Ca

ATP ase, H

pump 2. F-class

• locate on bacterial membrane , chloroplast and mitochondria

• pump proton from exoplasmic space to cytosolic for ATP synthesis

3. V-class

maintain low pH in plant vacuole

ATP-powered ion pumps generate and maintain ionic gradients across cellular membranes

Neel large energy: RBC need 50% ATP for Na/K pump; nerve and kidney need 25% for ion transport

Extracellular intracellular Extracellular intracellular

Muscle relaxation depends on Ca2+ APTase that pump Ca2+

from the cytosol into the sacroplasmic reticulum (SR)

In muscle cell, cytosol Ca2+ 10^-7 M (resting state) to 10^-6M (contraction)

Most intracellular Ca2+ storage in SR(10^-2M).

Ca2+ is released from the sarcoplasmic reticulum through Ca2+

release channels when the muscle contracts

Most cytosol Ca2+ transport into SR via Ca2+ ATPase pump.

Ca2+ pump comprises 90% of the sarcoplasmic reticulum membrane protein

Responsible for restoring the Ca2+ gradient (pumps it back into the sarcoplasmic reticulum

Operational model of the Ca²⁺-ATPase in the SR membrane of skeletal muscle cells

Higher Ca⁺²

Lower Ca⁺² Plays a major role in muscle relaxation

by transporting released Ca²⁺ back into SR

A single subunit protein with 10 transmembrane fragments Is highly homologous to Na,K-ATPase

10^-2

10^-6

Low affinity for calcium

Conformational change α-helix

Ca⁺⁺

ATP ADP + Pi

Ca⁺⁺

signal

calmodulin

endoplasmic reticulum

Ca⁺⁺

Ca⁺⁺-ATPase

Ca⁺⁺-release channel

outside of cell

ATP ADP + Pi

Ca⁺⁺

signal- activated

channel cytosol Ca⁺⁺-ATPase

Ca⁺⁺

Calmodulin-mediated activation of plasma membrane Ca2+ ATPase leads to rapid Ca2+ export → keep cytosolic Ca2+ very low

Allosteric activation

Calmodulin regulates the plasma membrane Ca2+ pump that control cytosolic Ca2+ concentration

ATP-powered ion pumps generate and maintain ionic gradients across cellular membranes

Extracellular intracellular

Na+/K+ ATPase maintain the intracellular Na+ and K+

concentration in animal cell

High low Low high Na+ transport out

K+ transport in

By Na+/K+ ATPase

Greatest consumer cellular energy

Sets up concentration & electrical gradients

Hydrolysis of 1 ATP moves 2K+ in and 3Na+ out against their concentration gradients

Na+/ K+ ATPase

(maintain the intracellular Na and K concentration in animal cell)

Higher affinity for Na+

Membrane potential Utilizes 30%

cells energy!

Four major domains:

Four major domains:

M M - - MembraneMembrane--bound domain, which is composed of 10 bound domain, which is composed of 10 transmembrane

transmembrane segmentssegments

NN- - NucleotideNucleotide--binding domain, where adenine moiety of binding domain, where adenine moiety of ATP and ADP binds

ATP and ADP binds

P P – – Phosphatase domain, which contains invariant Asp Phosphatase domain, which contains invariant Asp residue, which became

residue, which became phosphorylatedphosphorylated during the ATP during the ATP hydrolysis

hydrolysis A domain

A domain – – essential for conformational transitions between essential for conformational transitions between E1 and E2 states

E1 and E2 states

Na+/K+ ATPase

Na⁺- K⁺ Pump on the Plasma Membrane

K⁺ is 10 to 20 X higher inside animal cells than outside Na⁺ is 10 to 20 X higher outside animal cells than inside

These concentration gradients are maintained by the Na⁺ - K⁺ pump on the plasma membrane

– Pump operates as an antiporter, pumping K⁺ in and Na⁺ out

Transport cycle depends on autophosphorylation of the protein – Terminal phosphate of ATP is transferred to an aspartic

acid of the pump

• Ion pumps that autophosphorylate are called P-type transport ATPases

Na ⁺ - K ⁺ Pump on the Plasma Membrane

The Na⁺- K⁺ pump is electrogenic

– It generates an electrical potential (known as membrane potential) across the membrane

• Reason:

– Pumps 3 Na⁺ ions out for every 2 K⁺ ions it pumps in

– Thus the inside of the cell is negative relative to the outside

Electrogenic effect of the pump contributes only ~10% of the membrane potential

– remaining 90% is only indirectly attributable to the Na⁺- K⁺ pump (discussed later)

Effect of proton pumping by V-class ion pumps on H⁺ concentration gradients and electric potential gradients across cellular

membrane

V-class H+ ATPase pump protons across lysosomal an vacuolar membranes

Generation of

electrochemical gradient Electrochemical gradient

combines the

membrane potential and concentration gradient which work additively to increase the driving

force

Only transport H+

ABC Transporters

Largest family of membrane transport proteins

– 78 genes (5% of genome) encode ABC transporters in E coli – Many more in animal cells

– Known as the ABC transporter superfamily

They use the energy derived from ATP hydrolysis to transport a variety of small molecules including:

– Amino acids, sugars, inorganic ions, peptides.

ABC transporters also catalyze the flipping of lipids between monolayers in membranes

All ABC transporters each contain 2 highly conserved ATP- binding domains

Bacterial permease are ABC proteins that import a variety of nutrients from the environment

Structure of the E coil BtuCD protein, an ABC transporter mediating vitamin B12 uptake

ATP-binding cassette

ABC transporter

•2 T ( transmembrane ) domain, each has 6 α- helix form pathways for transported substance

•2A ( ATP- binding domain) 30-40% homology for membranes

i.e. bacterial permease

• use ATP hydrolysis

• transport a.a ,sugars, vitamines, or peptides

• inducible, depend on the environmental condition i.e. mammalian ABC transporter ( Multi Drug Resistant)

• export drug from cytosol to extracellular medium

• mdr gene amplified by drugs stimulation

• mostly hydrophobic for MDR proteins cancer cell resistant to drug mechanisms

Bacterial permeases are ABC proteins that import a variety of nutrients from the enviornment

About 50 ABC small-molecule pumps are known in mammals

A section of the double membrane of E. coli

Auxiliary transport system associated with transport ATPases in bacteria with double membranes

The transport ATPases belong to the ABC transporter supefamily

Examples of a few ABC proteins

Nature Structural & Molecular Biology 11, 918 - 926 (2004)

The Multidrug Resistance Protein (MDR)

ABC (ATP-binding cassette)

170 Kdalton P-glycoprotein that pumps hydrophobic drugs out of cells in a ATP-dependent fashion.

Uses the energy derived from ATP hydrolysis to export a large variety of drugs from the cytosol to the extracellular medium.

It reduces the cytoplasmic concentration of drugs and hence their toxicity. It therefore reduces the effectiveness of

chemotherapeutic drugs. It is overexpressed in some tumour cells. Need high concentration to killed cell.

It transports a wide range of chemically unrelated proteins including the anthracyclines, actinomycine D, valinomycin, and gramicidin.

The approximately 50 mammalian ABC transporters play diverse and important roles in cell and organ physiology

A typical ABC transporter consists of four domains – two highly hydrophobic domains and two ATP-binding catalytic domains

ATP binding leads to dimerization of the two ATP-binding domains and ATP hydrolysis leads to their dissociation.

Action of the Multi-drug Resistant Transport Protein

Mode of action of MDR1 involves flipping [flippase] or pumping of the lipid soluble drugs that typically have some positive charges across the membrane (ATPase side) to the exterior where the drug is released to the outside.

MDR1 is found in high activity in organs like liver and kidney that play a major role in the breakdown of drugs and other toxic substances but unfortunately reaches highly unregulated levels in corresponding tumor cells.

Structural model of E. coli lipid flippase, and ABC protein homologous to mammalian MDR1

ABC protein that transport lipid-soluble substrates may operated by a flippase mechanism

Proposed mechanisms of action for the MDR1 protein

Flippases

Lipids can be moved from one

monolayer to the other by flippase proteins

Some flippases operate passively and do not require an energy source Other flippases appear to operate

actively and require the energy of hydrolysis of ATP

Active flippases can generate membrane asymmetries

Certain ABC proteins “ filp” phospholipids and other lipid-soluble substrates from one membrane leaflet to opposite leaflet

Flippase model of transport by MDR1 and similar ABC protein.

Spontaneously

Diffuses laterally

Flips the charged substrate molecule

1. Hydrophobic portion of target molecule

spontaneously inserts itself into the inner leaflet

2. Molecule diffuses

laterally until it bumps into MDR

3. MDR “flips” the

molecule from the inner to outer leaflet (this step is energetically

unfavorable and requires ATP)

4. Molecule diffuses away and

5. Spontaneously moves out of the outer leaflet

Diseases linked with ABC proteins

1. ALD( X-link adrenoleukodestrophy)

defect in ABC transport protein( ABCD1)

located on peroxisome, used for transport for very long fatty acid;

absence ABCD1→ fatty acid →accumulate cytosol → cell damage 2. Tangiers disease

Dificiency in plasma ABCA1 proteins, which is used for transport of phospholipis and cholesterol

3. Cystic fibrosis

mutation of CTFR( cystic fibrosis transmenbrane regulator; a Cl^- transporter in the apical membrane of lung, sweat gland and pancrease)

licked it → did not resorption of Cl → taste salty→This leads to abnormalities in the pancreas, skin, intestine, sweat glands and lungs

腎上腺腦白質失氧症

脂蛋白缺乏疾病

囊狀纖維化

Polycystic kidney disease 多囊性腎病 這個病的病理是腎內生出很

多個小型的囊腫，這些囊腫 慢慢的長大，產生壓迫力，

使周圍的腎組織功能上產生 障礙，而致腎衰竭。 病徵.

多囊腎的病徵腎小管不大斷 擴大，令腎臟增大，以及影 響腎功能

PDK1 or PDK2 mutation Regulation of ion transport

Incorporation of FAs into membrane lipids takes place on organelle membranes

Fig 18-4 Phospholipid synthesis

Fatty acid didn’t directly pass membrane

Acetyl CoA ---Æ saturated fatty acid acetyl-CoA carboxylase

fatty acid synthase

Annexin V ; binds to anionic phospholipids

Æ long exposure of exoplasmic face of plasma memb.

Æ signal for scavenger cells to remove dying cells

Flippases move phospholipids from one membrane leaflet to the opposite leaflet

asymmetric distribution of phospholipids

senescence or apoptosis – disturb the asymmetric distribution

Phosphatidylserine (PS) and phosphatidylethanolamine: cytosolic leaflet exposure of these anionic phospholipids on the exoplasmic face – signal

for scavenger cells to remove and destroy

Annexin V – a protein that specifically binds to PS phospholipids fluorescently labeled annexin V– to detect apoptotic cells

flippase: ABC superfamily of small molecule pumps

In vitro fluorescence quenching assay can detect phospholipid flippase activity of ABCB4

Yeast sec mutant – at nonpermissive temp:

secretory vesicle cannot fuse with plasma

membrane – purify the secretory vesicles

(dithionite) Phospholipid flippase activity of ABCB4

有為的青年,伸個懶腰!

讓我們一起繼續前進 進入細胞生物學領域

另一個高深且符合我們的境界 也就是溫老師會考的範圍

也就各位研究所考試---可能考的地方之一 雖然這幾年國立大學研究所考得不多,

但勿恃敵之不來,恃吾有以待之

Nongated ion channels and the resting membrane potential

Gated: need ligand to activation; Non-gated: do not need ligand

Ion Channel (non-gate)

Generation of electrochemical gradient across plasma membrane i.e. Ca⁺ gradient

regulation of signal transduction , muscle

contraction and triggers secretion of digestive enzyme in to exocrine pancreastic cells

i.e. Na⁺ gradient

uptake of a.a , symport, antiport; formed membrane potential i.e. K+ gradient

formed membrane potential

Q: how does the electrochemical gradient formed?

Selective movement of Ions Create a

transmembrane electric potential difference

Depending on the type of the channel, this gating process may be driven by:

1. ligand binding (ligand-gated channels)

2. changes in electrical potential across cell membrane (voltage-gated channels)

3. mechanical forces acting on cellular components (mechanosensitive channels)

Ion gating Channel

Gated ion channels respond to different kinds of stimuli

Selective movement of ions creates a transmembrane electric potential difference

Ion no move → no membrane potential Ion move → create membrane potential

各位把它想 成細胞外大 量Na流入 內,結果膜 外電壓會出 現負

59mv

The membrane potential in animal cells depends largely on resting K⁺ channel

Many open K+ channel but few open Na+, Cl- or Ca2+ channels on animal membrane So major ionic movement across the membrane is K+; it form the inside out ward by

the K+ concentration gradient → creating an positive charge on the outside;

outward flow of K+ ions through these channels, also called resting K+ channels.

-59mv

-59mv

各位把它想 成細胞內大 量K流出去, 結果膜外電 壓會出現正 ,但膜內會 是負

Negative charge on intracellular organic anions balanced by K⁺ High intracellular [K⁺] generated by Na⁺-K⁺ ATPase

Large K⁺ concentration gradient ([K⁺]_i :[K⁺]_o ≈ 30)

Plasma membrane contains spontaneously active K⁺ channels ⇒ K⁺ move freely out of cell

As K⁺ moves out of cell, leaves negative charge build up ⇒ opposes further K⁺ exit At equilibrium, electrical force balances concentration gradient and electrochemical

gradient for K⁺ is zero (even though there is still a very substantial K⁺ concentration gradient)

Resting membrane potential = flow of positive/negative ions across plasma membrane precisely balanced

Membrane potential measured as voltage difference across membrane

For animal cells, resting membrane potential varies between -20 and -200 mV Negative value due to negativity of intracellular compartment compared to

extracellular fluid

Because K⁺ channels predominate in resting plasma membrane, resting membrane potential mainly due to K⁺ concentration gradient

Nernst equation permits calculation of membrane potential (V):

Potential difference exists across every cell’s plasma membrane.

– cytoplasm side is negative pole, and extracellular fluid side is positive pole Inside of cell negatively charged because:

– large, negatively charged molecules are more abundant inside the cell

– sodium potassium ATPase pump

– resting K+ ion channels (from in to out flow)

Ion channels contain a selectivity filter formed from conserved transmembrane a helices and p segments

Ion-selectivity filter

Transmembrane domain

Structure like but function different

Structure of resting K⁺channel from the bacterium

Streptomyces lividans

Ion channels contain a selectivity filter formed from conserved transmembrane α helices and P segment

Voltage-gated K+ channels have four subunits each

containing six transmembrane α helices

Ion channels are selective pores in the membrane

Ion channels have ion

selectivity - they only allow passage of specific

molecules

Ion channels are not open continuously,

conformational changes open and close

Smaller Na+ does not fit perfectly Each ion

contain eight water

molecules

Mechanism of ion selectivity and transport in resting K+ channel

EACH OF THE binding sites closely mimics potassium ions' octahedral

hydration shell, thereby minimizing the energy required to strip off their water coats.

Because of their smaller size, sodium ions don't fit in these binding sites as snugly and thus find the energetic cost of trading their water coat for a spot in the

selectivity filter too high.

In the vestibule, the ions are hydrated. In the selectivity filter, the carbonyl oxygens are placed precisely to accommodate a dehydrated K⁺ ion. The dehydration of the K⁺ ion requires energy, which is precisely balanced by the energy regained by the interaction of the ion with the carbonyl oxygens that serve as surrogate water molecules

Loss four of eight H2O

• 鈉離子通道（sodium channel）

0.3㎜×0.5㎜大小，但更重要的是其內表 面帶有極強的負電荷。這些負電荷主要 會把鈉離子拉向通道，這是因為脫水後 的鈉離子直徑要比其它離子小。

• 鉀離子通道

0.3㎜×0.3㎜的大小，但它們不帶有負電 荷 ，但是鉀的水合離子比鈉的水合離子 要小得多因此，體積小的水合鉀離子就 可以很容易地穿過這個較小的通道，而 鈉離子則不行 。

Patch clamps permit measurement of ion movements through single channels

effect effect V=I x R

Ion flux through individual Na+ channel

Novel ion channels can be characterized by a combination of oocyte expression and path clamping

Na+ entry into mammalian cells has a negative change in free energy

Transmembrane

forces acting on Na+

ions

Cotransport:

Use the energy stored in Na⁺ or H⁺ electrochemical gradient to power the transport of another substance

Symport: the transportd molecules and cotransported ion move in the same direction

Antiport: the transported molecules move in opposited direction

Depending on how many solute molecules are transported and in

what direction, carrier proteins are dubbed uniporters, symporters, or antiporters.

Coupling of Active Transport to Ion Gradients without energy, against gradient

In mammalian cells, Na⁺ electrochemical gradient is maintained across the plasma membrane by active

transport of Na⁺ out of the cell, using ATP as an energy source

– This electrochemical gradient provides the driving force for the active transport of a 2^nd solute

E.g. in intestinal and kidney cells, symport systems driven by the Na⁺ gradient are used to transport sugars and amino acids into the cells

Bigger the Na⁺ gradient, the greater the rate of solute entry

Only small hydrophobic molecules cross membrane by simple diffusion

Electrochemical gradient:

Membrane potential

Concentration gradient

Ion concentration gradients across the membranes

establishes the membrane electric potential

The differences in ion concentrations across the membrane establishes a

membrane electrochemical gradient

An electrochemical gradient combines the membrane potential and the concentration gradient

Operation Model for the two-Na⁺/one glucose symport

Glucose transport against its gradient in the epithelial cells of intestine

Na+ linked symporters import amino acids and glucose into animal cells against high concentration gradients

Carrier oscillates between state A and state B

Binding of Na⁺ and glucose is cooperative (binding of either ligand induces a

conformational change that enhances binding of the 2^nd

ligand)

Since Na+ higher in the

extracellular space (& very low inside), glucose

more likely to bind in A state Accordingly,

Na+ and glucose enter the cell (by an A to B transition) more often than they leave the cell

Result is net transport of Na+ and glucose into the cell

The Na+ gradient Is used to drive active transport of glucose

Na+ pumped out by an ATP-

driven pump

Three carrier proteins, appropriately positioned in the plasma membrane, function to transport glucose across the intestinal epithelium.

Without ion force

Increases PM area

Bacterial symporter structure reveals the mechanism of substrate binding

No 3-D Mammalian sodium symporter, it similar to bacterial sodium-amino acid transporter

Bind to sodium →conformation change → bind to amino acid → transport substrate

3-D structural of the two Na+

one leucine symporter

In cardiac or muscle, Ca2+ ↑ → contraction

Normal cytosol is 10000 fold of Ca2+ concentration than Cardiac (10^-6 → 10^-2 M) Cardiac muscles contain 3Na+/ 1 Ca2+ antiporter

Movement of three sodium is required to power the export of one calcium 3Na⁺ _outside + Ca⁺² _inside 3Na⁺ _inside + Ca⁺²_outside

maintenance of low cytosolic Ca ²⁺ concentration

i.e. inhibition of Na⁺/K⁺ ATPase by Quabain and Digoxin

raises cytosolic Na⁺

lowers the efficiency of Na⁺/Ca⁺² antiport

increases cytosolic Ca⁺²

( used in cogestive heart failure)

Na+ linked antiport Exports Ca+2 from cardiac Muscle Cells

More contraction

Electrochemical gradient

Normal condition

烏本 毛地黃素

Cotransporters that regulate cytosolic pH H₂ CO₃ H⁺ + HCO^-

H+ can be neutrolized by 1.Na⁺/HCO3^-/Cl^- antiport 2. Cabonic anhydrase

HCO3^- CO₂ +OH^- 3. Na⁺/H⁺ antiport

Carrier proteins in the plasma membrane regulate cytosolic pH (pH_i ) at about 7.2

There are two mechanisms by which this pH is regulated - H⁺ is transported out of the cell

Na⁺-H⁺ exchanger, an antiporter, couples the influx of Na⁺ to an efflux of H⁺

- HCO₃^- is brought into the cell to neutralize H⁺ in the cytosol Na⁺ -driven Cl^- - HCO₃^- exchanger uses a combination of the two mechanisms by coupling an influx of Na⁺ and HCO₃ to an efflux of Cl^- and H⁺

The activity of membrane transport proteins that regulated the cytosolic pH of memmalian cells changes with pH

A putative cation exchange protein plays a key role in evolution of human skin pigmentation

SLC245A5 a human

transporter regulated skin color

TEM of skin melanophores

Plant vacuole membrane

• pH 3—6

• Low pH, maintained by

V-class ATP-powered pump

pyrophosphate-hydrolyzing proton pump (PPi -powered pump)

The H+ pump inside → inside positive →

powers mover negative ion move inside; High positive inside

→antiport many ion and sucrose → inward

Numerous transport proteins enable plant vacuoles to accumulate metabolites and ions

More positive

Trans-epithelial transport

Import of molecules on the lumen side of intestinal

epithelial cells and their export on the blood facing sides

Transcellular transport of glucose from the intestinal lumen into the blood

1

2

3

4

Cl^- secretion

Basolateral Na+/ K+ ATPase generates Na+ gradient that drives the Symporter

Glucose + normal saline → co-transport for energy supply

[high]

microvilli

Mechanism of Action of Cholera Toxin

Acidification of the stomach lumen by parietal cells in the gastric lining

Parietal cells acidify the stomach contents while maintaining a neutral cytolic pH

P-class

1 2

3

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