Chapter 11 Cell Molec.txt

Passive Diffusion (Does not require integral membrane proteins), and Facilitated diffusion via channel proteins, Facilitated diffusion via channel proteins, active transport via pumps (these require intergral membrane proteins)

the net movement of like molecules or ions from an area of high concentraion to area of low concentration.

1. hydrophobicity of substance, 2.size of substance, 3.steepness in the concentration gradient, 4.lipid/protein composition of the bilayer, 5.charge differences between 2 compartments.

CAN: Small gases, nonpolar milecules, some small uncharged polar molecules-- CANNOT: Ions, large uncharged polar molecules (sugars), and charged polar molecules (A.A., ATP, Proteins, nucleic acids)

Using radioactivity or fluorescence. Fluorec=scence tag and use confocal microscop to visualize movement OR Pluse-Chase experiment.

-Purify a transport protein of interest from cells using detergents -mix the protein with pure phospholipids and add to water -phospholipids spontaneously form liposomes when added to water -the transport protein will be incorporated into this liposome (free from other proteins). (Liposome is artificially prepared vesicles made of lipid bilayer ex. by sonication)

Cells use energy (ATP) to pump substances from an area of low concentration to an area of high concentration. Active transport is needed because of control- cells have to strictly control ion concentration within and outside the cell. cells want to bring in nutrients from the enviornment even if the cytoplasm has high concentrations of that nutrient. (another ex. is getting rid of toxin)

1. pH- inside the cell is kept at 7.2, 2.Na+- extracellular has ~10x higher conc of Na+ than the cytosol. 3. K+ cytosol has ~20x higher concentrations of K+ than the extracellular fluid. 4. Ca+ the cytosol has very little Ca+ (1000x less than the extracellular fluid)

P-Class Pumps, F-Class, V-Class, ABC pumps.

P Class Pumps transport onyl ions and contain 2 subunits- alpha and beta. Alpha- catalytic subunit(binds to ATP and transports the ions), Beta- regulatory subunit.the alpha subunit gerts phosphorilated urign the process, phosphorilation induces a change in pump structure = allows for transport. Examples include: EUK plasma membrane Na+/K+ and Ca2+ pumps, H+/K+ pump in the stomach (H out of cell/K into cells), H+ pump of bacteria fungi and protist, Ca+ within muscle cells-

SR Ca2+: 1. cytoplasmic portion of the alpha subunit binds to 2 Ca ions and a molecule of ATP 2.ATP is hydrolyzed into ADP+P 3. This phosphorylation causes a conformational change in the pump 4.Ca ions come off and go into the LUMEN 5.the phospate is removed and the protein goes back to its original comformation. Na+/K+: 1. 3Na+ ions and ATP bind to the alpha subunit 2.ATP hydrolyzed and P is transferred as before 3.conformational change allows Na+ to be released to the outside 4.2 K+ ions bind to other sites in the alpha subunit 5.phosphate is removed, protein reverts back to original conformation 6.the bound K+ ions are released into the cytosol (3Na+ out/2K+ in).

ATP powered pump that pumps protons, found in bacterial PM, mitochondrial inner membrane, and chloroplast thaylakoid membranes. Functions in the production of ATP following electron movement through the ETC. AKA ATP synthases

Pumps protons only. very complex structure (compared to P-class). Primarily function to pump protons into animal cell lysosomes/late endosomes and plant vacuoles. Also found in the membrane of osteoclasts (bone remodel/breakdown).

contain 2 large domains each composed of multiple subunits. V1=hydrophilic cytosolic domain (ATP hydrolysis). V0= hydrophobic transmembrane domain.

ATP is bound and hydrolyzed by the V1 subunit. Provides the power to pump H+ through the V0 domain. Nothing gets phosphorylated like in P-Class. A Cl- must be pumped in (via different pump) every time an H+ is transported into the lysosome. Must keep the charge balance neutral.

Addd a particle labeled with a fluorecent dye that changes color when exposed to different pHs.

very diverse class (100 diff membranes). Pumps various types of substances -sugars, a.a., toxins, etc. Diff types pump diff substances. All contain 2 transmembrane domains (that form the passageway) and 2 cytosolic domains that function to bind ATP. Found all over bacterial and EUK plasma membranes.

Bacteria contaion numerous ABC pumps called permeases in their PM. -Use energy from ATP hydrolysis to pump in nutrients such as a.a., sugars, and peptides against steep concentration gradients. -Bacteria regulate expression of these permeases depending on their needs and how many nutrients are in the enviornmen. Mammals have MDR1 (multidrung-resistance transport protein) -pumps various toxins/drugs from the cytosol to the outside (keeps conc in cytoplasm very low) -such chemicals are typically small hydrophobic molecules that freely diffuse through the bilayer.

Channel proteins create a hydrophylic passageway through which water, ions, and small organic molecules move across the membrane-Can be a single polypeptide folded in such a way to create a passageway (e.g., beta barrel)-Can be a complex of different polypeptides that together form a passageway � Most channel proteins have open and closed conformations-Some of these open and close in response to stimuli (ligand binding, movement, or light) or changes in voltage across the membrane -Called gated channels-Opening and closing of other channelsare unaffected by these factors-Called nongated channels

Water has the ability to diffuse through channel proteins called aquaporins -Diffusion of water = osmosis� Water diffusion follows the same general rules of diffusion-Water moves from an area of high water concentration to an area of low water concentration-When is there a difference in water concentration?-Never really, but there can be differences inthe amount of �FREE� water molecules -Free water are those molecules that are capable of diffusing (those not associated with an ion or something else)-A solution with high solute concentrations will have low levels of free water and vice versa (Note: All solutes are created equal when it comes to osmosis)-Thus, water will diffuse from an area of Low solute to an area of High solute�

If you add a cell to a solution containing more or less solutes than what is found in the cytoplasm, water will diffuse. -Some terms when comparing 2 environments:- Hypotonic�The environment containing less solutes- Hypertonic�The environment contain more solutes- Isotonic�When solute concentration is equal in the 2 environments

Plant/fungal/bacterial cells-Contain a rigid cell wall that allows for cell swelling, but prevents cell lysis-Swelling of the cell (due to entry of water into a vacuole) creates pressure called TURGOR PRESSURE (this prevents more water from coming in)� Animal cells-Do not contain a CELL WALL -Constantly ship out certain ions into the extracellular fluid in an attempt to make that fluid isotonic with respect to the cytoplasm (no water influx)� Protozoans-Contain a special structure called a CONTRACTILE VACUOLE that is constantly collecting extra water from the cytoplasm and shipping it out

Complex of 4 identical subunits, each containing 6 membrane-spanning alphahelices-In 3-D, these alpha helices form a CENTRAL PORE that allows for the flow of water molecules- HYDROGEN BINDING provides specificity-Hydrophilic R groups extend in the middleof the channel and perfectly hydrogen bond with water molecules-Water moves single file through it-Different cell types have different levels of aquaporins(e.g. kidney cells)-Diabetes insipidusand aquaporin2 mutations

Nongatedion channels are extremely SELECTIVE -K+ channels only allow for the diffusion of K+. Na+ channels only allow for the diffusion of Na+.

Most contain 4 identical subunits arranged around a central pore-Each subunit contains a loop that extends into the pore (provide ion selectivity)-Each loop perfectly takes the place of the water shell that forms around the ion

Two sides of the PM end up with different NET CHARGES -The plasma membrane of most eukaryotic cells is more negative on the inside face than on the outside face-This difference in charge between the 2 sides of the membrane is called the MEMBRANE POTENTIAL or voltage -The membrane potential of a normal resting cell is -70 mV. 1) The cytoplasm contains a high concentration of LARGE ANIONIC MOLECULES (e.g. proteins) that are unable to exit the cell 2) K+is allowed to move DOWN ITS CONCENTRATION GRADIENT and diffuse out of the cell(Na+and Ca2+are not allowed to diffuse into the cell as easily)-This results in more + outside (more �inside) -How does K+get back into the cell eventually (Na+/K+ pump)?Note: Plant cells maintain this -70 mV mem. potential by constantly shipping H+ out of the cell via P-class pumps

1) Patch-clamp technique -Designed to measure the inward or outward flow of ions through a SINGLE ion channel complex -Can be used for any type of ion channel (gated or non-gated) -Utilizes 2 electrodes a) One is directly attached to a PATCH OF MEMBRANE that should only contain a few ion channels -This patch electrode contains a SALT SOLUTION specific for the channel to be studied (this will be the source of ions) b) One that is inserted into the CYTOPLASM (intracellular electrode)- The patch-clamp maintains a constant membrane potential -Example: For every Na+leaving the patch electrode, the intracellular electrode injects an electron into the cytoplasm (keeps the cytoplasm neutral) -It measures how many e-are injected 2) OOCYTE EXPRESSION-Frog eggs contain FEW OR NO ION CHANNELS -Can inject the mRNA encoding a newly-discovered ion channel into a frog egg-The ion channel protein will be made and inserted in the PM -Use the patch-clamp technique to determine which ion it transports, how long it stays open, what induces it to open (is it gated), etc.

1) VOLTAGE �Changes in membrane potential trigger the opening of ion channels in neuronal and muscular tissue 2) LIGANS �Chemicals such as neurotransmitters and hormones bind directly to the ion channel and cause them to change conformation 3) Activated G-proteins� Binding of a ligandto a receptor often activates a cytosolicprotein called a G protein. The activated G protein leaves the receptor, binds to a nearby ion channel, and causes it to open 4) Light�Ion channels in cells of the inner eye open in response to light(via a chemical called rhodopsin) 5) Many others�Ion channels in the inner ear open in response to physicalmovement (vibrations)

All have 4 polypeptides or 1 polypeptide with 4 separate domains that arrange in a cylinder with a central pore-All have VOLTAGE SENSING ALPHA-HELICES that contain lots of lysinesand arginines(+ charged). In the resting (closed) state of the channel, the + charged ahelices are situated closer to the CYTOPLASM (b/c they are attracted to the �charges)-The gate is closed and no ions move through-When the membrane has a SMALL DEPOLARIZATION (the cytoplasm becomes + with respect to the outside), the ahelices move up towards the OUTSIDE-Induces a conformational change in the channel -->Opens up -->ions flow. -Soon after ion flow begins, the ahellicesgo back to their original position-A piece of the channel called the CHANNEL INACTIVATING SEGMENT will then block the pore, preventing further ion flow-It will remain there until the membrane is REPOLARIZED (inside -, outside +)-Like the nongatedchannels, these are very specific for a given type of ion-Have similar types of loops that selectively guide specific ions through the channel opening

Cell body(soma) �Contains the nucleus and other organelles -DENDRITES�Receives signals from the environment or from other neurons -Axon�SEND AN ELECTRICAL IMPULSE TO ANOTHER CELL(neuron, gland, or muscle) -Axon termini contact these other cells

1) A SMALL DEPOLARIZATION of the membrane occurs as a result of other ionchannels that open in response to ligandsand various other stimuli-Small amounts of Na+ diffuse into the cell-The more stimuli, the longer these channelsare open, the more Na+ comes in-Inside of the membrane at this location isnow DEPOLARIZED (inside + with respect to theoutside)2) The depolarization of this patch of membrane triggers the opening of VOLTAGE GATED Na+ CHANNELS, which allow even more Na+ to flow into the cell -This depolarizes more of the membrane, which triggers more voltage-gated Na+ channels to open (like a positive feedback loop)-This continues until the gate closes or the excess of internal + chargesrepel further movement of cations inward 3) The rush of Na+ into the cytoplasm (and subsequent depolarization of the membrane) eventually causeS VOLTAGE-GATED K+ CHANNELS to open -Allows K+ to flow OUT OF CELL (along with the flow through the nongatedchannels)-Eventually enough K+ leaves the cytoplasm so that the membrane potential is restored (inside -, outside +)4) Na+/K+ pump sends Na+ back out and K+ back in (restores normal conc. gradients)

� NICOTINIC ACETYLCHOLINE RECEPTOR -A prototypical ligand-gated ion channel -This receptor is involved in the transfer of an electrical impulse from a neuron to a muscle cell (it is a receptor and channel in one) Activation of the NAR: 1) The arrival of an action potential to the end of an axon triggers the secretion of acetylcholine(type of neurotransmitter) into the space between the neuron and muscle cell 2) Acetylcholine moves across this NEUROMUSCULAR JUNCTION binds to the outside of NARs located on the muscle cell PM -This binding leads to a change in receptor conformation, which allows Na+ TO FLOW INTO THE CELL (creating a localized depolarization) 3) An action potential is then initiated in the muscle -->leads to contraction

Opening of some ion channels is regulated by the action of a CYTOSOLIC PROTEIN called a G protein (peripherally associated with the PM) -Binding of a ligand to its receptor signals for the recruitment and ACTIVATION of a G protein -Activated G protein leaves the receptor and binds to a nearby PM ION channel -It will bind to the channel and induce a conformational change -The channel OPENS and allows for the diffusion of some ion -This type of signaling is a little delayed (always a delay between ligand addition and channel opening)

Transporters (or carrier proteins) are integral membrane proteins that "grab" various ions/nutrients, undergo a conformational change, and transport the cargo across a membrane� Transporters fall into 1 of 2 general categories 1) UNIPORTERS� Bind to a single substance and transport it across the membrane (down conc. grad) 2) Cotransporters�Bind to multiple substances and transport them TOGETHER- Symporters�Transport 2 or more substances in the same direction- ANTIPORTERS�Transport 2 or more substances in the opposite directions

� Common theme with cotransporters- One substance is moving down its concentration gradient -This movement is energetically favorable (releases energy) -Released energy is used to transfer a second substance AGAINST its conc.gradient (same end result as active transport pumps)

Most uniportersexist in 2 conformational states (see GLUT1 below -glucose uniporter)-One has the cargo binding site facing the outside of the cell, the other has thebinding site facing the cytoplasm-Binding of cargo to this site initiates a SHIFT TO THE OTHER STATE-that releases the cargo to the other side- Movement is dependent upon the conc. gradient-When glucose conc. is high outside of the cell, GLUT1 will transport it in-When glucose conc. is high inside of the cell, GLUT1 will transport it out

1) Na+/glucose symporter-Na+ is allowed to move into the cell (down its conc. gradient). The ENERGY RELEASED from that movement is used to bring in glucose with it (against its gradient)-Neither will be transported without the other-Present in cells where glucose movement is unfavorable (e.g. small intestine)

2) Na+/amino acid symporter-Same principle as described for #1 3) Na+/Ca+ antiporter - Na+ brought in (favorable), Ca2+ shipped out (unfavorable) 4) pH controlling antiporters