Lecture 4- transport across membranes

Lecture 5- transport across membranesnow playing: go outsidethe cultsAlberts, Chapter 12

matters arisingmembrane transport goalsby the end of this topic, you should know why membrane transport is important to cell biologyknow what kinds of molecules enter a cell easily and which must be actively moved in or out know what proteins allow transport and what aspects of their protein structure are important to their function and selectivity compare the properties of channels vs active transporters and know how they relate to energy be able to describe how disruptions in membrane transporters contribute to diseases or mutant animal behaviors be able to describe the key transporters and ions required for maintaining resting potential and generating an action potential3transport across membranes - overview Simple diffusion of uncharged molecules (down a gradient) Osmosis Ion channels (regulated, but diffusion down a gradient) Facilitated diffusion Pumps (active transport UP a gradient) Energy storage in electrochemical gradients Defective transport leads to disease Membrane potentials and nerves4how can you tell the topology?

N-term 1 50 100AA C-termhydrophobicity

NC(and you understandthat actual proteins aremore complex than myline drawings)

NCrest of the cellrest of the celldoesnt clarify which side is cytoplasmic5a test for membrane topologyadd trypsinpurify membranesSDSpageexperiment 1trypsin is an enzyme that breaks proteins into very small pieces,too small (generally) to see in an SDS-PAGE gel

trypsin cannot get into a cell thus transmembrane and internal proteinsare protecteda test for membrane topologyadd trypsinpurify membranesSDSpageexperiment 1add trypsinpurify membranesor permeabilizeSDSpageexperiment 2return to membrane transportalready in progressif you put a cell in water

what happens?how do you interpret the result?

Large uncharged polar molecules like glucose, sucrose, amino acids or IONS are not permeable and must be transported by proteins.membranes selectively allow molecules acrosssome molecules penetrate the lipid bilayer:those that are soluble in oilsor very small moleculesThese may also go through PORESsemi-permeable(in and out)10 Plasma membrane encloses the cell Must let in nutrients and signals Must export trash Must exclude undesireable materials Must be selectiveactive vs. passive transportPassive transport: from area of high to low concentrationActive transport: from area of low to high concentration

11active vs. passive transport

H20H20second law of thermodynamics: entropy spontaneously increases -DG is energetically favorable things move down a gradientthat also means - you need to do work/expend energy to add order to a system or to move up a gradient 12energetics of solute movementThe free energy change that occurs when a solute diffuses across a membrane is dependent on the difference in concentration on the two sides of the membrane.For an uncharged substance - non-electrolyte moving into the cell:G = RT ln Ci/Co or G = 2.303 RT log10 Ci/CoG is the free energyR is the gas constantT is the absolute temperature in degrees KelvinCi/Co is the ratio of [solute] inside to [solute] outside the membrane

13H2O can diffuse quickly through membranes H2O moves more quickly through membranes than dissolved ions or small solutesOsmosis: movement of H2O from an area of low solute concentration to an area of higher solute concentration

can get hypotoniclysisghost14Hypertonic vs. hypotonic vs. isotonic

Plant cells maintain turgor pressure15H2O enters/exits cells through specialized pores called aquaporins H2O molecules pass through one by one Channel wall is positively charged and binds to negatively charged O; thereby disrupting H bonds that link H2O molecules together

HOhttp://nobelprize.org/nobel_prizes/chemistry/laureates/2003/chemanim1.mpgsimple diffusion doesnt account for all H2O movement across membranes16 Channel lined with hydrophobic amino acids H2O molecules pass through one by one 1billion per second

17 Channel lined with hydrophobic amino acids H2O molecules pass through one by one 1billion per second

facilitated diffusion (transport) of glucose through membranes via transportervarious polar solutes aretransported by this mechanism, down a [gradient]Most cells contain a glucose transporter that facilitates the diffusion of glucose from the blood stream into the cell to be used for energyTransport 102-105 molecules/secfacilitative(passive) transporter

18Transport depends on concentration gradient Polar solutes (sugars, amino acids) Transporter undergoes conformational change after binding solute Can transport in either directionPhosphorylation of glucose within the cell maintains a unphosphorylated glucose gradient

Insulin hormone produced by endocrine cells of the pancreas - it maintains blood sugar levels.1) at low insulin levels few transporters are on the cell surface.

2) Insulin stimulates the exocytosis of the glucose transportersSome adult type 2 diabetes results from deficiency in the Glut 4 glucose transporter.

glucose uptake is controlled in part by regulation of its transporter on the cell surface19Glucose is the major energy source Elevated blood glucose can be lethal All cells have glucose transporters; however, muscle and fat cells take up the most gluocse Increases in blood glucose (after eating) stimulate production of insulin Insulin (produced by pancreatic islet cells) induces the uptake of blood glucose into cells When insulin levels are low, glucose transporters (GLUT4) reside intracellularly; high blood glucose levels induce transporters to translocate from cytoplasm to plasma membrane

What if a cell already has high glucose?

Symport same directionAnti port different directions 20

At room temperature ~ 25CG = (1.4 Kcal/mole) log10 Ci/Co

in this example, Ci/Co will be less than 1 and G will be negative and a net INFLUX of solute is thermodynamically favored. (log Ci logC0)CoCi101Cellif G is positive, it represents the energy required to pump the solute across the membrane if G is negative you do not need to put energy in.For diffusion of a solute to the outside of the cell Co becomes the numerator in the concentration ratio term where the solute is going is the numerator. it is thermodynamically favored to move down a concentration gradientENERGY IS STORED BY DIFFERENCES IN CONCENTRATION

21Na+ 145mMK+ 5Mg++ 1-2Ca++ 1-2H+ pH 7.4

Cl- 110Na+ 5-15mMK+ 140Mg++ 0.5Ca++ 10-4H+ pH 7.2

Cl- 5-15typical mammalian cellmajor differences in ion concentrations in and outside the cellextracellularlots of biomolcules have negative charges Excess extracellular Na+ and intracellular K+ are balanced by other molecules22Na+ 145mMK+ 5Mg++ 1-2Ca++ 1-2H+ pH 7.4

Cl- 110Na+ 5-15mMK+ 140Mg++ 0.5Ca++ 10-4H+ pH 7.2

Cl- 5-15typical mammalian cellmajor differences in ion concentrations in and outside the cell store energyextracellularlots of biomolcules have negative charges Excess extracellular Na+ and intracellular K+ are balanced by other molecules23

it is thermodynamically favored to move down a concentration gradient which can allow something else to move up its concentration gradient24

transport of glucose through membranes via active transportersintestinal and kidney cells take up glucose from the intestinal lumen or kidney tubules where the glucose concentration is low and actively transport it across the membrane using energy from coupled import of Na+ ion --- an example of symport

25Na+ 145mMK+ 5Mg++ 1-2Ca++ 1-2H+ pH 7.4

Cl- 110Na+ 5-15mMK+ 140Mg++ 0.5Ca++ 10-4H+ pH 7.2

Cl- 5-15typical mammalian cellabout these ions - why doesnt everything go to equilibrium? how does the cell make gradients?extracellular(note that overallthe cell is neutralwith respect to charge)lots of biomolcules have negative charges Excess extracellular Na+ and intracellular K+ are balanced by other molecules26

how does a cell make gradients?hard workCells USE ENERGY in different formsto establish gradientsmovement against a concentration gradient occurs by active transportActive transport requires energy (ATP hydrolysis)Na+150 mM15 mMCa2+10-3 M10-7 M pumps mediate active transport drive a given ion in only one direction28movement against a concentration gradient occurs by active transportPumps are critical: maintain low pH inside lysosomes maintain low pH inside the stomach

29

key example: Na+/K+ ATPase pumpwe will return to this but first more ways ions movemany kinds of ion/tranport systems bacteriorhodopsin is a light-activated proton pumpa photon of light changes the structure of retinal, starting a relay that results in proton release on the extracellular side then the retinal is re-protonated

Archea31charged ions move through gated channels small ions (K+, Na+, Ca2+, Cl-) cant diffuse through lipid bilayers necessary for nerve impulses, muscle contraction, etc. most ion channels are selective for specific ionswhen the gate is open, ions flow down their concentration gradient-no other energy supplied(fast! 10 million K+/sec)

what would happen if there was no gate?

33who/what are the gate keepers? voltage-gated channels: open based on differences in membrane potential ligand-gated channels: open based on a ligand binding to the channel (conformational change) mechanosensory gated channels: open in response to force or other stimuli

34conformational changes close the channel

this is due to changesin voltage more on that next time

K+ channel of bacteria (KcsA channel) hydrophobic alpha helices span membrane (integral membrane protein) tetramer selectivity filter only permits K+ ions to bind K+ is normally hydrated (O); Filter provides Os (electronegative); Only K+ ions fit into the selectivity filter mammalian channels are very very similar ancient origin

36a fly mutant for the K+ channelhttp://www.pnas.org/content/suppl/2005/02/22/0406164102.DC1/06164Movie1.movEpisodic Ataxia Type-1 (EA-1) is considered the human equivalent of the Shaker mutation in Drosophila. EA-1 is a rare autosomal dominant neurological disorder that results in uncoordinated movements (ataxia) that may last from several seconds to hours. Genetic linkage studies have identified the gene responsible for EA-1 as the Shaker-related Kv1.1 gene.

a cool, hot channel

TRPV is gated by ligands or by heat (& pain)

they allow cations suchas Ca++, Mg++through

TRP family members have diverse activators and initiate signal transduction cascadesTransient Receptor Potential Vanilloiden.wikipedia.org/wiki/TRPVyou discovered a new channel!you name your new channel Ch 303 - HDyour observation: it multimerizes and folds in such a way that there is a very small pore lined with amino acids with a positive or partially positive charge

you try a patch clamp experimentmeasures ion flow by measuring electrical current

the Na+/K+ ATPase pump (1) Na+ binds to the transport protein on the inside of the cell with high affinity ATP is hydrolyzed and the released P binds to the transport protein binding of P changes the transporters configuration and affinity for Na+ ; Na+ is released to the outside of the membrane

this is a P pump

41flashback: for reversible chemical reactions, forward and reverse reaction rates are equal at equilibriumfor the reaction A + B C

at equilibrium, kfor[A] [B] = krev[C]

and, = = Keq kforkrevkfor krev [C] [A][B] for a given concentrationof reactants/products, you can calculate Keq.if Keq > 1, the forward reaction is favoredk is the rate constant,and the forward rate is determined by kfor [A][B]where [ ] denotes Molarconcentrationsbut this does not describe reaction ENERGYall cellular rxns are reversibleat equilibrium, the free energy change from reactants to products goes to zero42an aside on affinitymost of the time proteins are associated with something else in the cell in a complex (C) via non-covalent bondsin protein (P) -ligand (L) interactions, affinity is described by a dissociation constant Kd:

Kd = [P] [L] [C]

when amount of protein (alone) = the amount of protein in a complex (ie, HALF of the total protein is in a complex with the ligand), Kd=[L]

if the protein and ligand have high affinity, very little ligand is required to get a complex(in moles) K+ binds to the pump P dissociates causing the pump resumes its original conformationthis lowers K+ binding affinity K+ diffuses into the cytoplasm

the Na+/K+ ATPase pump (2)44critical elements of Na+/K+ pump Pumps ions AGAINST the concentration gradient Hydrolysis of ATP is required (P-type pump: hydrolysis of ATP results in phosphorylation of the transport protein) Transport protein must have a higher binding affinity for Na+ inside the cell and a lower binding affinity for Na+ outside of the cell Transport protein must have a higher binding affinity for K+ outside of the cell and a lower binding affinity for K+ inside the cell Different affinities are achieved by phosphorylating the transport protein Pump is necessary to maintain steep gradient required for nerve and muscle cell activation. 45long-awaited crystal structures of the Na+ K+ PumpOgawa H, Shinoda T, Cornelius F, Toyoshima CCrystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabainProc. Natl. Acad. Sci. U. S. A. v106, p.13742-13747Shinoda T, Ogawa H, Cornelius F, Toyoshima CCrystal structure of the sodium-potassium pump at 2.4 A resolutionNature v459, p.446-450Morth JP, Pedersen BP, Toustrup-Jensen MS, Sorensen TL, Petersen J, Andersen JP, Vilsen B, Nissen PCrystal structure of the sodium-potassium pumpNature v450, p.1043-1049review: Structural biology:Ion pumps made crystal clearDavid C. GadsbyNature 450, 957-959(13 December 2007)doi:10.1038/450957a

Na+ 145mMK+ 5Mg++ 1-2Ca++ 1-2H+ pH 7.4

Cl- 110Na+ 5-15mMK+ 140Mg++ 0.5Ca++ 10-4H+ pH 7.2

Cl- 5-15typical mammalian cellmajor differences in ion concentrations in and outside the cell store energyextracellular(note that overallthe cell is neutralwith respect to charge)lots of biomolcules have negative charges Excess extracellular Na+ and intracellular K+ are balanced by other molecules47many ATP-powered pumps Na+/K+ pump is only in animal cells H+/K+ pump in the stomach (pumps acid into the stomach; pump translocates to plasma membrane after eating) H+ proton pump in plants is important for import of solutes and control of pH ABC (ATP-binding cassette) superfamily of pumps present in bacteria through mammals pump ions, sugars, peptides, polysaccharides, proteins! Defects in pumps, transporters, or channels often lead to disease48

model of an ABC transporter49multi-drug resistance (MDR) tumor cells become resistant to chemotherapy MDR-1 protein is part of a pump (ABC transporter) that pumps toxic materials out of cells MDR-1 expressed in normal liver and kidney to export toxic molecules (e.g. bile, urine) MDR-1 transports many drugs/toxic materials BUT in cancer cells: MDR-1 gene is amplified and over-expressed so chemotherapy drugs that diffuse through the cancer cell membrane are pumped out50

diseases linked to ion channels51case study: Cystic Fibrosis (CF) abnormal secretion of fluid by epithelial cells of the airways CF patients make thick, sticky mucous that stays in airways cilia cant move properly so bacteria remain infections and inflammation destroys lung function lethal 1/25 people of Northern European origin are genetic carriers; 1/2500 infants affected autosomal recessive52Cystic fibrosis patients have a defect in an ABC transporter (cystic fibrosis transmembrane conductance regulator, CFTR) with several functions forms a cAMP-regulated Cl- channel transports bicarbonate (HCO3-) ions blocks Na+ channel activates several chloride/bicarbonate transporters53why do the mutations lead to disease?Cystic Fibrosis patients commonly have a particular amino acid change in the CFTR protein, DF506CFTR is a ABC transporter that acts at the plasma membrane by transporting Cl- and suppresses Na+ ion channel function in epithelial cells

to study this mutation, you clone both normal and DF506 mutant copies of the gene and add a GFP tag. you then express the protein in cells and look for the GFP expression. you see:next time:membrane potentials,using the energy you have stored, neurotransmitters, diseases related to membrane transport