Lodish Berk Kaiser Krieger Scott Bretscher Ploegh Matsudaira

MOLECULAR CELL BIOLOGY SIXTH EDITION CHAPTER 11 Transmembrane Transport of Ions and Small Molecules 2008 W. H.Freeman and Company Copyright 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 arent required for transport

Sugars diffuse very slowly so proteins are involved in transport.

Charged molecules are virtually impermeable.

Only small hydrophobic molecules cross membraneStudies 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.

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

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 sizeThe 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

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

Overview of membrane transport proteins 1. All transmembrane proteins 2. Some transport has ATP binding sites 3. Move molecules uphill () 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

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.

Passive transport driven by: Facilitated Diffusion Concentration gradient(affects both uncharged and charged solutes)

+Electrical gradient(affects only charged solutes)

Electrochemical gradientCopyright 2009, Dr. Salme Taagepera PhD. All rights reserved.

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

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.

Facilitated Diffusion

Free DiffusionA. Non-channel mediated lipids, gasses (O2, CO2), water B. Channel mediated ions, charged molecules

Facilitated diffusionCarrier 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

Several feature distinguish uniport transport from passive diffusionNeed a carrier protein Unique features for Uniport transport: 1. Higher diffusion rate for uniport than passive diffusion. 2. Transported molecules never enter membrane and Irrelevant () to the partition coefficient. (did not cross membrane) 3. Transport rate reach Vmax 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

GLUT: glucose transport

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

Glucose transporter (GLUT): Facilitated Diffusion

Families of GLUT proteins(1-12) Highly homologous in sequence, and contain 12 membrane-spanning helices. Different isoforms different cell type expression, and different function GLUT2: express in liver cell ( glucose storage) and 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: ???

Mammalian glucose transportersName Glut1 Glut2 Glut3 Glut4 Glut5 Glut7 Glut8 Tissue distribution all fetal and adult tissues hepatocytes, pancreatic -cells intestine, kidney widely distributed skeletal muscle, heart, adipocytes Proposed function basal glucose transport transepithelial transport from and to the blood basal glucose transport mostly in brain insulin-dependent transport

intestine, lesser amounts few others fructose transport gluconeogenic tissues preimplantation blastocyst mediates flux across endoplasmic reticulum embryonic insulindependent 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

GLUT1 deficiency syndrome

Glucose

GLUT1 is responsible for transporting glucose across the bloodbrain 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)Copyright 2009, Dr. Salme Taagepera PhD. All rights reserved.

Transporter proteins (membrane protein) can be enriched within artificial membraneChloroform 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 CB concentration > CA

Osmotic pressure =RT( CB-CA)

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 (): 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

Water draw is equal inside and outside 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

Aquaporins increase the water permeability of cell membrane Water cross membrane is very lower Water cross membrane via specific channel- aquaporins

Expression of aquaporin by frog oocytes increases their permeability Egg move to hypotonic environmentInjection aquaporin mRNA

control

Aquaporin 1 erythrocyte Aquaporin2

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

Water channel protein ( aquaporin) Tetrameric protein

6 -helices for each subunit

2-nm-long water selective gate 0.28nm gate width Highly conserved arginine and histidine in the gate H2O for HO bonding with cystein

Aquaporins are membrane water channels that play critical roles in controlling the water contents of cells. Water crosses the hydrophobic membrane either by simple diffusion or through a facilitative transport mechanism mediated by these specialized proteins. These protein channels are widely distributed in all kingdoms of life, including bacteria, plants, and mammals. Important in osmotic regulation, acting to prevent bursting of the cells whenever there are changes of the exterior salt concentration.

ATP powered pump1. P- class 2, 2 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 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 cytosolic for ATP

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.

inside

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

inside

ATP powered pump 1. P- class 2, 2 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

Extracellular intracellular

Extracellular intracellular

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

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 cellsPlays 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

Higher Ca+2

10-6Low affinity for calcium

Lower Ca+2

-helix

Conformational change

Calmodulin regulates the plasma membrane Ca2+ pump that control cytosolic Ca2+ concentrationCalmodulin-mediated activation of plasma membrane Ca2+ ATPase leads to rapid Ca2+ export keep cytosolic Ca2+ very low

Ca++ signal Ca

calmodulin

Ca++-release channel++

endoplasmic reticulum

ATP

Ca ATP

Ca++-ATPase ++ ADP + Pi Ca++ ADP + Pi

Allosteric activationsignalactivated channel Ca++

cytosol outside of cell

Ca++-ATPase

Na+/K+ ATPase maintain the intracellular Na+ and K+ concentration in animal cell ATP-powered ion pumps generate and maintain ionic gradients across cellular membranes

Na+ transport out K+ transport in By Na+/K+ ATPase

Extracellular

intracellular

High Low

low high

Na+/ K+ ATPase (maintain the intracellular Na and K concentration in animal cell)Greatest consumer cellular energy Sets up concentration & electrical gradients Hydrolysis of 1 ATP moves 2K+ in and 3Na+ out against their concentration gradientsHigher affinity for Na+

Membrane potential

Utilizes 30% cells energy!

Na+/K+ ATPase Four major domains: M - Membrane-bound domain, which is composed of 10 transmembrane segments N- Nucleotide-binding domain, where adenine moiety of ATP and ADP binds P Phosphatase domain, which contains invariant Asp residue, which became phosphorylated during the ATP hydrolysis A domain essential for conformational transitions between E1 and E2 states

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

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

Na - K Pump on the Plasma Membrane

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)

V-class H+ ATPase pump protons across lysosomal an vacuolar membranesOnly transport H+ Effect of proton pumping by V-class ion pumps on H+ concentration gradients and electric potential gradients across cellular membrane Generation of electrochemical gradient Electrochemical gradient combines the membrane potential and concentration gradient which work additively to increase the driving force

ABC TransportersLargest 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 ATPbinding domains

Bacterial permease are ABC proteins that import a variety of nutrients from the environment ATP-binding cassette

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

Bacterial permeases are ABC proteins that import a variety of nutrients from the enviornment About 50 ABC small-molecule pumps are known in mammals ABC transporter 2 T ( transmembrane ) domain, each has 6 - helix form pathways for transported substance 2A ( ATP- binding domain) 30-40% homology for membranesi.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

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

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.

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 ProteinMode 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.

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

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

Proposed mechanisms of action for the MDR1 protein

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

FlippasesLipids 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

Flippase model of transport by MDR1 and similar ABC protein.1. Hydrophobic portion of target molecule spontaneously inserts itself into the inner leaflet Molecule diffuses laterally until it bumps into MDR MDR flips the molecule from the inner to outer leaflet (this step is energetically unfavorable and requires ATP) Molecule diffuses away and Spontaneously moves out of the outer leaflet

2.

Spontaneously Diffuses laterally

Flips the charged substrate molecule

3.

4.

5.

Diseases linked with ABC proteins1. 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 Cltransporter in the apical membrane of lung, sweat gland and pancrease)licked it did not resorption of Cl taste saltyThis leads to abnormalities in the pancreas, skin, intestine, sweat glands and lungs

Polycystic kidney disease . PDK1 or PDK2 mutation Regulation of ion transport

Incorporation of FAs into membrane lipids takes place on organelle membranesFatty acid didnt directly pass membraneAcetyl CoA -------- saturated fatty acid acetyl-CoA carboxylase fatty acid synthase Annexin V ; binds to anionic phospholipids long exposure of exoplasmic face of plasma memb. signal for scavenger cells to remove dying cells

Fig 18-4 Phospholipid synthesis

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

Phospholipid flippase activity of ABCB4

Yeast sec mutant at nonpermissive temp: secretory vesicle cannot fuse with plasma membrane purify the secretory vesicles

(dithionite)

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

,! ------ , ,

Nongated ion channels and the resting membrane potentialGated: 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

Ion gating ChannelDepending 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)

Gated ion channels respond to different kinds of stimuli

Selective movement of ions creates a transmembrane electric potential difference

59mv

Na ,

Ion no move no membrane potential Ion move create membrane potential

-59mv -59mv K, ,

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

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 cells 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 helices and P segmentIon channels contain a selectivity filter formed from conserved transmembrane a helices and p segments Ion-selectivity filter

Structure like but function different

Structure of resting K+channel from the bacterium Streptomyces lividans

Transmembrane domain

Voltage-gated K+ channels have four subunits each containing six transmembrane 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

Mechanism of ion selectivity and transport in resting K+ channel

Each ion contain eight water molecules

Smaller Na+ does not fit perfectly

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

sodium channel 0.30.5 0.30.3

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 gradientIn 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 2nd 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

The differences in ion Electrochemical gradient: concentrations across the membrane establishes a membrane electrochemical gradient Membrane potential Ion concentration gradients across the membranes establishes the membrane electric potential

Concentration gradient

An electrochemical gradient combines the membrane potential and the concentration gradient

Na+ linked symporters import amino acids and glucose into animal cells against high concentration gradients Operation Model for the two-Na+/one glucose symport

Glucose transport against its gradient in the epithelial cells of intestine

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 2nd 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 ATPdriven 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 bindingNo 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

Na+ linked antiport Exports Ca+2 from cardiac Muscle CellsIn 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 calcium3Na+ outside+ Ca+2 inside Normal condition maintenance of low cytosolic Ca 2+ concentration i.e. inhibition of Na+/K+ ATPase by Quabain and Digoxin 3Na+ inside+ Ca+2outside

raises cytosolic Na+Electrochemical gradient

lowers the efficiency of Na+/Ca+2 antiport

increases cytosolic Ca+2 ( used in cogestive heart failure)

More contraction

Carrier proteins in the plasma membrane regulate cytosolic pH (pHi) at about 7.2Cotransporters that regulate cytosolic pH H2CO3 H+ H+ + HCO-

can be neutrolized by 1.Na+/HCO3-/Cl- antiport 2. Cabonic anhydrase HCO3CO2+OH-

3. Na+/H+ antiport 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 36 Low pH, maintained by V-class ATP-powered pump

Numerous transport proteins enable plant vacuoles to accumulate metabolites and ions

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

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

Glucose + normal saline co-transport for energy supply

3[high]

1

2

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

4

microvilli

Cholera toxin activated Cl- secretion

Mechanism of Action of Cholera Toxin

Parietal cells acidify the stomach contents while maintaining a neutral cytolic pHAcidification of the stomach lumen by parietal cells in the gastric lining

3 1 P-class 2

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