Lipids, Membranes and Transport Outline Biological Membranes o Framework of the membrane is the phospholipid bilayer o Phospholipids are

e amphipathic (forms bilayer) molecules Hydrophobic (water-fearing) faces in Hydrophilic (water-loving) faces out o Membranes contain Proteins and carbohydrates Fluid Mosaic Model o The membrane is considered a mosaic of lipid, protein and carbohydrate molecules o Membrane resembles a fluid- lipids and proteins move around relative to each other Lateral movement across membrane Membrane Proteins o Integral or Intrinsic membrane proteins Transmembrane Proteins Region physically embedded in the hydrophobic region of bilayer Lipid anchored Protein Noncovalent attachment of a lipid to amino acid side chain within a protein o Peripheral membrane (extrinsic proteins) Noncovalently bound to regions of integral membrane proteins that project out from membrane or bound to polar head groups of phospholipid o Hydrophilic Head- loves water o Hydrophobic Tails- hates water

Hydrophobic Amino acids

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Lipids, Membranes and Transport Outline 1.Alpha-helix 2. Multiple alpha helices

3. series of alpha helices 4. 1 layer of bilayer

5. Lipid anchored

Approximately 25% of all genes Encode membrane proteins o Membranes are extremely important- biologically and medically Membranes are Semifluid o Fluidity Individual molecules remain in close association but have ability to move within the membrane o Semifluid Most lipids can rotate freely around their long axes and move laterally within the membrane leaflet Flipflop of lipids from one leaflet to the opposite leaflet does not occur spontaneously Flippase requires ATP to transport lipids from one leaflet to another

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Lipids, Membranes and Transport Outline Factors affecting Fluidity o Length of fatty acyl tails Hydrophobic tails- the shorter the tail, the more fluid it is o Presence of double bonds in the acyl tails Big factor in fluidity Double bond creates a kink in the tail, making it more difficult to interact with nearby tails and making the bilayer more fluid o Presence of Cholesterol Tends to stabilize membranes Depends on temperature Cold-rigid Warm-fluid

Head

Loves water

Tails

Hates water 2 C=C bonds

Head

1 C=C bonds

1 C=C bonds 0 C=C bonds Saturated

Saturation o Saturated is when there are no C=C double bonds o Unsaturated is when there are C=C double bonds Cholesterol Stabilizes Membranes o High Temperatures Lipid becomes fluid Cholesterol becomes more rigid o Low Temperatures Lipid becomes more rigid Cholesterol becomes more fluid

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Lipids, Membranes and Transport Outline Lateral Transport Experiment o Mouse and Human cells fused together o Added a florescent label antibody and saw o 0 C- Cold temperature Protein unable to move laterally and remains on one side of the cell o 37 C- Higher temp Movement observed, the protein is on both sides of the cell Some Integral Membrane Proteins have restricted movement o 10%-70% of membrane proteins may be restricted in their movement Anchored, attached to cytoskeleton or extracellular matrix o Membrane proteins may be attached to molecules outside of the cell, so the network of proteins that forms the extracellular matrix

Transmembrane Protein

How to study Membranes o TEM (Transmission Electron Microscopy) Thin section sample stained with heavy metal dyes, gives electron dense, binds to polar bits since metal has a charge Train track looking membrane bilayer (polar heads dyed)

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Lipids, Membranes and Transport Outline o FFEM (Freeze Fracture Electron Microscopy Specialized form of TEM, can be used to analyze the interiors of phospholipid bilayers Sample is frozen in liquid Nitrogen and fractured with a knife Leaflets separate into P face (protoplasmic) and E face (extracellular) Provide 3D detail about membrane protein form and shape Using FFEM- Branton in 1966 was able to conclude that membranes are formed from phospholipid bilayers, with proteins intercalated within

Synthesis of Membrane Components o In Eukaryotes, Cytosol and endomembrane system work together to synthesize most lipids o Process occurs at cytosolic leaflet of smooth ER o Fatty acid building blocks made via enzymes in cytosol or taken into cells from food

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Lipids, Membranes and Transport Outline All phospholipids are synthesized in the Smooth ER Transfer of Lipids to other Membranes o Lipids in ER membrane can diffuse laterally to nuclear envelope o Transported via vesicles to Golgi, lysosomes, vacuoles, or plasma membrane (blebbing) o Lipid exchange proteins- extract lipid from one membrane for insertion in another o 2 Phospholipids into 1 Membrane Protein Synthesis o Most transmembrane proteins are directed to ER membrane o From the ER, membrane proteins can be transferred via vesicles to other regions of the cell o Trans-membrane (expand layer) proteins are translated directly into the phospholipid bilayer of the rough ER Stretches of 20 hydrophobic amino acids (universal for transmembrane proteins) Tends to form alpha-helical structure Length of region is 20 amino acids long How transmembrane proteins are translated

Protein finished being translated

Protein traffiking o Proteins destined for any part of the endomembrane system or secretion from the cell are translated directly into the ER Anything membrane bound is directly translated into the ER membrane itself- even some non-membrane proteins also

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Lipids, Membranes and Transport Outline o How proteins are sorted once in the endomembrane system is reliant on the physical properties of lipid vesicles

- Like a lava lamp- things can bud off and go back and forth, not unidirectional

-Most things can go back and forth with vesicles, not unidirectional

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Lipids, Membranes and Transport Outline Protein Sorting to the ER

Docks with protein in Rough ER to translate

Signal recognition protein (SRP)

Translated into ER lumen

Transmembrane Protein- 20 amino acids, forms alpha helice

Polypeptide chain- inside ER lumen

Protein Sorting to the Golgi o From ER to Golgi o Uses membrane proteins V-Snare and T-Snare to merge protein-containing vesicles to the correct target o Certain proteins are classified as V-Snare or T-Snare Proteins that form matrix (cage)

Golgi

Pinches off (Blebs off) Rough ER Inside ER Lumen 8|Page

Attaches to T-Snare

Lipids, Membranes and Transport Outline Protein Sorting in the Golgi (continued) o Cargo protein, targeted to the golgi, binds to a cargo receptor protein in the ER membrane. Coat proteins on the outside aid in the formation of vesicle buds, which will contain the appropriate cargo. In the vesicle membranes are also V-snares.proteins embedded in the membrane which target the vesicle to the correct site. Upon approaching the target (Golgi), T-snare proteins within the golgi membrane bind with the v-snares, causing the membranes to fuse, and the cargo protein to be released into the golgi. Coat Proteins cause the membrane to Bleb Off

SARE Proteins allow vesicles to join together- (Direct mechanism) o 2 Nerves

- Transmembrane Proteins

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Lipids, Membranes and Transport Outline

Spontaneously Happens
-Forms one larger unit -T-Snare attaching to Golgi

Glycosylation o Process of covalently attaching a carbohydrate to a protein or lipid Glycolipid- Carbohydrate to lipid (sugar stuck to fats) Glycoprotein- Carbohydrate to protein (sugar stuck to protein) o Proteins ER to Golgi o Can serve as recognition signals for other cellular proteins o Plays a role in cell surface recognition o Protective effects Cell coat or glycocalyx- carbohydrate rich zone on the cell surface shielding cell

Clicker Question o The bond that attaches Carbohydrates to Lipids and Proteins in the Plasma Membrane Covalent Bond Because were transferring to O or N

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Lipids, Membranes and Transport Outline Protein Glycosylation o N-Linked In ER & Golgi Attachment of Carbohydrate (sugars) to Nitrogen (N) atom of asparagine side chain o O-Linked Only in Golgi Addition of sugars to Oxygen (O) atom of Serine or Threonine side chains Both have hydroxyl groups (OH), so sugars get added on there N-Linked Glycosylation o Carbohydrate (sugars) to N atom of asparagine side chain o In ER & Golgi o Dolichols are previously made in the ER o Transfer to target Asp sequence o Commonly found in membrane proteins

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Lipids, Membranes and Transport Outline O-Linked Glycosylation o Occurs only in Golgi o Links sugar chains to the O in Ser or Thr side chains o Important for production of proteoglycans Mucous Extracellular matrix o Transfers sugar group onto OH of Ser and Thr Endocytosis and Exocytosis o Transport larger molecules such as proteins and polysaccharides, and even very large particles o Usually larger things gets endocytosed or exocytosed Ex. Load of Insulin excreted from Pancreatic cell o Exocytosis From inside cell to outside cell Material inside the cell packed into vesicles and excreted into the extracellular matrix o Endocytosis Plasma protein invaginates or folds inward to form a vesicle that brings substances into the cell Receptor-mediated endocytosis Pinocytosis- Small molecules in extracellular environment are brought into the cell Used for absorption of extracellular fluids Phagocytosis- Eat Endocytose bacteria Engulfing solid particles by the cell membrane Defense mechanism- to remove cellular debris- immune response Exocytosis o Inside cell to Outside the cell

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Lipids, Membranes and Transport Outline Endocytosis o Into the Cell, receptor-mediated o Blebs off into the cell o Guided by cytoskeleton (carries cargo)

Phospholipid Bilayer Barrier o Serves as a barrier to hydrophilic molecules and ions due to hydrophobic interior and hydrophilic outside o Rate of diffusion depends on chemistry of solute and its concentration o Gasses and a few uncharged molecules can passively diffuse across o Ions and large polar molecules diffuse slowly
- Things that can get thru freely- Hydrophobic molecules. Gasses, small molecules -This is how cells breathe Diffusion -Diffuse thru membrane freely

Small polar molecules diffuse thru somewhat slow, uncharged.

Large molecules, more charged and harder to diffuse, cant go thru bilayer

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Lipids, Membranes and Transport Outline Membrane Transport o Selectively permeable plasma membrane Outside environment cant dictate inside environment o Structure ensures Essential molecules enter Metabolic intermediates remain Waste products exist Movement Across Membrane o Passive transport doesnt require an input of energy- down or with gradient Passive Transport Diffusion of a solute through a membrane without transport protein Facilitated Diffusion Diffusion of a solute through a membrane with the aid of a transport protein o Active transport requires energy- up or against gradient

-Simple diffusion -No transport protein -Down/with gradient, no energy required Ex.) O2, CO2

-Diffusion with Aid of transport protein -Down/with gradient, no energy required Spontaneous, favorable

-Up/Against gradient -Requires energy Non-spontaneous, unfavorable G = (+)

G = (-)

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Lipids, Membranes and Transport Outline Active Transport o Movement of a solute across a membrane against its gradient from a region of low to higher concentration (Low Concentration High Concentration) o Energetically unfavorable, non-spontaneous o Requires input of Energy to drive movement o Primary Active Transport Uses a pump- directly uses energy to transport solute o Secondary Active Transport Very common Uses pre-existing gradient to drive transport of solute Relying on concentration gradient- ATP is not always used

Low Conc.

High Conc. Cells Maintain Gradients o Living cells maintain a relatively constant internal environment different than outside the cell o Transmembrane gradient Concentration of a solute is higher on one side of a membrane than the other o Ion electrochemical gradient Both an electrical gradient and chemical gradient

Glucose
Transmembrane Gradient Conc. Of glucose is higher outside than it is inside the cell

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Lipids, Membranes and Transport Outline

Ion Electrochemical Gradient -Electrical and Chemical gradient -More Na+ outside the cell and higher Positive charge outside the cell

Tonicity o Comparison of solute concentrations across a barrier o In relation to the cell Isotonic- Equal amounts of solute inside and out of the membrane (and equal amounts of water) Hypertonic- Solute concentration is higher on one side of the membrane (and water concentration lower) Hypotonic- Solute concentration is lower on one side of the membrane (and water concentration is higher) -Solute concentration outside the cell is Isotonic to the inside of the cell

-Solute Concentration outside the cell is Hypertonic to the inside of the cell

-Solute concentration outside the cell is Hypotonic to the inside of the cell

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Lipids, Membranes and Transport Outline Osmosis o The movement of water towards a solute o Water diffuses through a membrane from an area with more water to an area with less water o If solutes cannot move, water movement can make the cell shrink or swell as water leaves or enters the cell o Osmotic Pressure- The tendency for water to move into any cell o Animal cells must maintain a balance between extracellular and intracellular solute concentrations to maintain their size and shape o Crenation-shrinking in a hypertonic solution- (can kill cells this way) Aquaporins o Channel proteins that move water across membrane o Discovery of Aquaporins CHIP28 Water passively diffuses across plasma membrane Certain cell types allow water to move across the plasma membrane at a much faster rate than would be predicted by passive diffusion Peter Agre and colleagues first identified a protein that was abundant in RBCs and kidney cells, but not found in many other cell types Striking difference was observed between frog oocytes that expressed CHIP28 versus the control CHIP28- renamed Aquaporins Transport Proteins o Transmembrane proteins that provide a passageway for the movement of ions and hydrophilic molecules across membranes o 2 classes bases on movement type Channels- forms a pore to shuttle thru Transporters- Carrier proteins, transforms and changes shape to pass through

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Lipids, Membranes and Transport Outline Transporters o Carriers o Conformational change transports solute o Principal pathway for the uptake of organic molecules Sugars, amino acids, nucleotides

o Types of Transporters o Uniporter- Single molecule/ion moves in and out of the cell o Symporter/Cotransporter- 2 or more ions or molecules transported in same direction o Antiporter- 2 or more ions or molecules transported in opposite directions Glucose Transporters o Some glucose transporters are uniporter o Ex.) Blood brain barrier (BBB), glucose is transported across endothelial cells of small blood vessels into astrocytes o Uniporters that transport Glucose down its concentration gradient o Glucose transporters undergo conformational changes that result in a reorientation of their substrate binding sites across membranes

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Lipids, Membranes and Transport Outline Channels o Form an open passageway for the direct diffusion of ions or molecules across the membrane o Most are gated o Ion Channels

Hydration of Ions o Ions in aqueous solution o Influences their flux through Transmembrane pores o Salts dissolved in water form hydrated ions o The hydrophobicity of lipid bilayers is a barrier to movement of hydrated ions across cell membranes Ion channels Dehydrate Ions o All ion channels dehydrate ions o Hydration shell must be removed for ions to be selectively shuttled through a channel o Allow for the rapid selective transport of ions across membranes o Dehydration of Ions costs energy, whereas hydration of ions frees energy o Almost every ion channel is gated Electrochemical gradients o Across the cell membrane generate the Membrane Potential o Nernst Equation- used to calculate the membrane potential as a function of ion concentrations o Cells maintain a negative resting membrane potential with the inside of the cell slightly more negative than the outside o Membrane potential is a prerequisite for electrical signals and for directed ion movement across cellular membranes

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Lipids, Membranes and Transport Outline

K+ Potassium Channels o Catalyze selective and rapid ion permeation o Function as water-filled pores that catalyze the selective and rapid transport of K+ ions o Complex of 4 identical subunits, each of which contributes to the pore (tetramer)

o Selectivity filter- evolutionary conserved structure o K+ channel selectivity filter catalyzes dehydration of ions, which confers specificity and speeds up ion permeation o How it works:

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Lipids, Membranes and Transport Outline

6 Packets
P0,P1,P2,P3,P4,P5 2 Configurations 1,3 or 2,4

Keeps moving K+ up one rungevery other, so it pushes up, its staggered every other one. -Because positive charges repel, repulsion moves cells up the channel

P5 is where the K+ hydrated ion enters and in here, it dehydrates the K+ ion and allows it to fit specifically into P4 to P0 Gating of K+ Channels o Gating means being able to shut if off (plug it up) o Different gating mechanisms define functional classes of K+ channels o Distinct from the selectivity filter o Regulated by the membrane potential

Pushes up by repulsion Opens when K+ is present 21 | P a g e

Pinch closed at bottom Moves paddles, inverts

Able to close around inside of channel

Lipids, Membranes and Transport Outline Aquaporin channels o Selective water transport occurs through Aquaporin Channels o Have to dehydrate ions to move them through o Allow rapid and selective water transport across cell membranes o Tetramers of four identical subunits, with each subunit forming a pore Aquaporin Selectivity Filter o Has 3 major features that confer a high degree of selectivity for water Size restriction Electrostatic repulsion Water dipole orientation o Water molecules have to line up single file based on dipole moments o Tetomer- forms like an hourglass, thin in the middle Action Potentials o Electrical Signals o Depend on several typs of ion channels o Enable rapid communication between cells Nerve cells and muscle cells use these o Na+, K+, Ca2+ currents are key elements of action potentials, but not the only ones o One way that cells communicate with each other (rapidly) o Uses electrochemical gradients o Resting Potential- The electrical potential that exists due to a difference in charge across a membrane. The charge thats maintained so the signal can be transmitted Resting Potential- forces K+ against its gradient + charge outside, - inside

Voltage gated channels-Triggers gated ion channels to open, triggers others to open up as well Action potentials are mediated by ion currents- depends on ion concentrations 22 | P a g e

Lipids, Membranes and Transport Outline Action Potentials o Membrane depolarization is mediated by the flow of Na+ ions into cells through voltage dependent Na+ channels o Repolarization is shaped by transport of K+ ions through several different types of K+ channels o Electrical activity of organs can be measured as the sum of action potential vectors Na+ Gradient o Used for getting lots of stuff into the cell If we want to get something into the cell we can use Na+ ion to couple it, to get it inside t o Transmembrane Na+ gradient is essential for the function of many transporters o Plasma membrane Na+ gradient is maintained by the action of the Na+/K+-ATPase o Used for Glucose and other ions o Na+/K+-ATPase is an ion driven pump

ATP-driven Ion Pumps Generate Ion Electrochemical Gradients o Na+/K+-ATPase Actively transport Na+ and K+ against their gradients by using the energy from ATP hydrolysis (to ADP) 3 Na+ exported for 2 K+ imported into the cell Antiporter- different directions Electrogenic pump- export 1 net positive charge (3 ions out, 2 ions in)

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Lipids, Membranes and Transport Outline Reaction cycle is described by the Post-albers scheme, which proposes that the enzyme cycles between two fundamental conformations

Model for transport by the Na+/K+-ATPase 1. 3 Na+ ions bind a. ATP to ADP, and the protein now has a phosphate 2. Conformational change of molecule (protein) 3. Release 3 Na+ ions outside the cell and its opened up to allow K+

4. 2 K+ binds into pockets, protein gets dephosphorylated

5. Protein closes outside of cell portion, protein gets phosphorylated with ATP 6. Releases K+ ions into the cell 24 | P a g e Starts all over again

Lipids, Membranes and Transport Outline Na+ gradient formed by Na+/K+-ATPase powers the transport of: o Examples Na+/H+ and Na+/HCO3-cotransporters Regulates cytostolic and extracellular pH Na+/Glucose transporter Intestinal cells absorb glucose Na+/K+/Cl- cotransporter Regulates intracellular Cl- concentrations Na+/Mg2+ exchanger Transport Mg2+ outside of the cell Na+/Ca2+ exchanger Major transport mechanism for removal of Ca2+ from the cytosol of excitable cells

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