Cell biology ch. 8

Over secretion of acid can lead to heartburn. Several mechanisms of treating the symptoms and cause are known.

1. Prilosec OTC or Previced are widely used and prescribed drugs that prevent heartburn by inhibiting the stomach's H+/K+-/ATPase. 2. Zantac, Pepcid, and Tagamet work instead by blocking a receptor on the surface of the parietal cells, preventing the cell from becoming activated by the hormone. 3. Tums and Rolaids work by neutralizing the acid (H+) in the stomach after it has been made.

Facilitating transport proteins share properties of enzymes

1. Transport proteins are specific for the solutes they transport. There is probably a specific binding site analogous to an enzyme's active site. 2. Transport proteins can be saturated with solute, so a maximum transport rate occurs when all binding sites are occupied with solute. 3. Transport proteins can be inhibited by molecules that resemble the solute normally carried (similar to competitive inhibition in enzymes) **not only moving glucose also a.a.s & others!!

Example mechanisms of gating:

1. Under resting conditions with a negative charge across the membrane, the channel is closed due to one of the alpha helices being misplaced so that no ions can move through it. 2. A positive membrane potential causes the helix to rotate in such a way that the channel opens and ions are free to pass. 3. Once the pore is open, a few thousand ions pass through the channel per millisecond. 4. If the membrane voltage returns to resting the channel will close due to movement of the S4 helix. 5. Once the inactivating particle moves into the channel the protein cannot be immediately reopened even if the membrane voltage is favorable.

A membrane may be permeable to a given solute either...

1. because the solute can pass directly through the lipid layer 2. because that solute can traverse an aqueous pore that spans the membrane

transport protein oscillates between 2 conformation

1. high affinity for Na+ with binding sites oriented towards the cytoplasm 2. high affinity for K+ with binding sites oriented towards the cell's exterior

Factors that affect the simple diffusion of a molecule:

1. partition coefficient: a measure of the polarity (or nonpolarity) of a substance... ► the ratio of its solubility in a nonpolar solvent such as octanol or veggie oil, to that in water when the oil and water are mixed. ► The more soluble a molecule is in the membrane, the faster it will penetrate the membrane. 2. size: rate of movement through a membrane depends on the compounds size ► if two molecules have approx. equivalent partition coefficients, the smaller will penetrate the membrane more rapidly than the larger. ► very small uncharged molecules penetrate a membrane very quickly ◙ such as O2, CO2, NO, H2O are thought to slip between adjacent phospholipids

There are overall two categories for the movement of substances through a membrane:

1. passively by diffusion 2. actively by an energy coupled transport process

Think of the plasma membrane as having a dual function:

1. prevent loss of cytosol into the environment 2. allow exchange of substances across the membrane with the environment

There are several specific methods by which substances move across membranes:

1. simple diffusion-- through the lipid bi-layer 2. pore --simple diffusion through an aqueous protein-lined channel 3. Carrier (facilitated diffusion) 4. active transport

Diffusion is a spontaneous process in which a substance moves from a region of high concentration to a region of low concentration...eventually eliminating the concentration gradient. Two qualifications must be met before a nonelectrolyte (charge must also be considered when moving ions) can diffuse passively across a plasma membrane.

1. substance must be present at higher concentration on one side of the membrane than other 2. the membrane must be permeable to the substance

Model of Facilitated Diffusion: Alternating Conformation Model of glucose transport:

1. transport protein remains in place in the membrane and translocates solute by alternating between 2 conformations. 2. transport protein might bind to solute in one conformation and deposit it on the other side of the membrane in another conformation 3. the solute's binding and release may trigger the transport protein to revert to its original conformation Example: Glucose Uptake via the glucose permease, GLUT1 ► concentration outside the cell is at 5m, concentration within the cell is much lower ► glucose has a low permeability coefficient the power that drives this is concentration gradient.

the K+ concentration inside and outside a mammalian cell is about:

100 mM inside, while outside the cell is only about 5mM. ► this favors the efflux of K+ from the cell

the Na+ concentration is about what inside/outside of cell?,

150 mM outside the cell and 10-20mM inside the cell. ► this favors influx of Na+ ions into the cell

how many different forms of GLUT are there?

6

the blood cell's trick:

??? allows the cell to carry more oxygen then it should be able to.

Larger polar molecules such as sugars amino acids, and phosphorylated intermediates have poor membrane penetrability.,

► As a result, the lipid bi-layer provides an effective barrier that prevents these important molecules diffusing out of the cell. ► these molecules require a protein be present in the membrane to let them into/out of the cell

The high-energy electrons associated with NADH and FADH2 are transferred (dumped) to a series of specific electron carriers that constitute the ETC (Electron Transport Chain) located in the inner mitochondrial membrane.

► Complex I, II, III, and IV ► These protein complexes will use energy from the elections to establish a H+ gradient within the intermembrane space. ► electrons are accepted then passed on at a slightly lower energy to the next carrier protein in line ► lowest energy electrons are ultimately passed to molecular oxygen from Complex IV The stored H+ gradient (potential energy) will power the very endergonic process of synthesizing ATP via secondary or indirect active transport by the F0/F1 complex.

Question: What type of empiracal evidence would support the idea that an ion is required to move large molecules across a membrane?

► Figure 8-10, pg 212

Often the function of a cell or even a particular biochemical pathway is accomplished through the action of several proteins working in concert. This can be demonstrated in a simple example and in more complex ones.Simple example: Consider the physiologic activity of the intestine.

► In the lumen, enzymes hydrolyze high molecular-weight polysaccharides into simple sugars, which are absorbed by the epithelial cells that line the intestine. ► glucose moves across the apical plasma membrane against a concentration gradient by Na+ cotransport, i.e. by symport. ► the needed Na+ gradient is maintained by active transport by the Na+/K+-ATPase pump in the basal and lateral membranes of the cells ► finally the glucose molecules move into the blood stream by facilitated diffusion through a glucose transporter (glucose permease)

OR

► The movement of ions through the channel results in movement of the cytoplasmic N- terminus domain, termed the inactivating particle, into the open pore causing it to be inactivated.

Why make this gradient in the first place?

► The subsequent facilitated diffusion of Na+ back across the membrane drives the intake of glucose, and other molecules ► this is often termed indirect active transport or secondary active transport

Total ionic permeability for a particular ion determined by:

► Total number of membrane channels that allow that ion to cross. ► The ease with which an ion can go through a single channel. ► Easier to measure relative ion permeabilities than absolute permeabilities.

TCA: Tricarboxilic Acid Cyle

► a.k.a. Citric Acid cycle, a.k.a. Kreb's cycle ► cyclic pathway that occurs in the matrix of mitochondrion ► acetyl CoA is substrate for TCA ◙ acetyl CoA is formed via β-oxidation pathway in the mitochondrial matrix & in the peroxisome or ◙ by the Transition Reaction as pyruvate crosses the mitochondrial membranes generates1NADHandreleases1CO2

The velocity of glucose uptake can be saturated.

► at high [glucose] all the transporters are occupied, i.e., Vmax ► Km for D-glucose for this transporter is about 1.5mM ◙ the Km represents a measure of the affinity of the transporter for glucose ► the Km for L-glucose is >3000mM ◙ important concept: unlike simple diffusion, facilitate diffusion is sterospecific...will readily bind D-glucose but not L-glucose ► transporter will also bind other 6-carbon sugar molecules that are similar in structure to D- glucose: ◙ These sugars can competitively inhibit the uptake of D-glucose into erythrocytes. --D-mannose Km = 20mM --D-galactose Km = 30mM !!! transporters can easily recognize these sugars (D) rather than L-glucose.

Mitochondrion Structure:

► enclosed by a double membrane ◙ space between membranes is termed the intermembrane space ◙ site of H+ storage during ETC ► outer membrane is fairly smooth ► inner membrane is very convoluted and rich in embedded proteins ◙ infoldings of membrane are termed cristae ◙ large surface area ◙ ETC enzymes are present here ► solution within inner membrane is termed the matrix ◙ site of TCA pathway ◙ site of mitochondrial gene expression ◙ site of mitochondrial genome replication

Mitochondrion Basics:

► found in nearly all eukaryotic cells ► not part of endomembrane system ► are semiautonomous, meaning they grow and divide within the cell and generate some of the proteins that function there, and take up others from the cytoplasm ◙ have their own DNA and ribosomes ► number per cell varies with cell's function and metabolic activity ► 1μm in diameter X 1-10μm in length but shape and size are very dynamic ◙ It has been proposed that they are actually much larger than detailed but so convoluted within the cell that it is difficult to discern true shape.

If the glucose molecules can diffuse in and out of the cell based on concentration...how does the cell prevent reaching equilibrium? How does it maintain a concentration gradient?

► gradient of high glucose levels outside the cell is maintained immediately phosphorylating glucose upon its entering the cell ◙ this effectively removes that glucose molecule from the transporters' gradient potential preventing them moving back through the channel ► This is a common trick used by cells to lower the interior cell concentration to encourage facilitated diffusion!!!

There are other diseases that have been identified as a similar transporter problem...

► in a kidney disease, CYSTINURIA, the transport proteins for cysteine and other amino acids is missing from the membrane

How are such gradients made and maintained?

► not simple diffusion... ► more than facilitated transport...

Glycolysis:

► pathway by which monosaccharides (such as glucose) are broken down ► occurs in cytosol of eukaryotic cells and in prokaryotes

The amount of energy associated with electrons removed in an oxidation depends on the orbitals they occupied in the oxidized molecule. The energy of the removed electrons is measured and expressed as a relative potential or voltage by comparison with an arbitrary standard.:

► the energy of electrons removed from hydrogen in the reaction: H2--- > 2H+ + 2e- ► the potential applied to these electrons is 0.000V. All other potentials, called reduction-oxidation potentials or E', are measured and assigned a value with respect to this standard.

open channels

► usually open. Allow ions to move back and forth across the membrane without regulation

One result of such ion movements is a net negative charge on the inside of most plasma membranes.

► usually ranges between -15mV and -100mV ► termed membrane potential in nonexciteable cells (those other than neurons and muscle) ► termed a resting potential in neurons and muscle cells

Sodium-glucose symport:

Cells lining our intestine are able to take up molecules like glucose or amino acids from the lumen into the cells even when the cellular concentration is higher than the lumen concentration. ► this seems like active transport, however, the driving exergonic reaction here is the use of the Na+ gradient established by the Na+/K+ pump Conformational change in this protein causes it to have a high affinity for glucose when open to the extracellular face if Na+ is present to bind simultaneously. ► the result is movement of glucose against a concentration gradient without direct hydrolysis of ATP

Side Note:

Diffusion is the principle uses in dialysis, a very valuable lab technique. As shown at right, a protein solution with contaminating high salt can be rid of the salt by placing the solution in a semi-permeable membrane termed dialysis tubing and exposing the tubing to several changes of the desired buffer.

K+ channel gene mutations

Problems with heart beat polarization

in which con. does GLuT1 have low affinity for its ligand??

T2 (when its open to inside of cell)

Example of Transport Proteins and Medicine:

The epithelial lining of the stomach contains a H+/K+-ATPase which secretes a solution of concentrated acid (up to .16NHCl) into the stomach chamber. ► in resting state, these pumps are situated in the membranes of cytosolic vesicles ► when food enters the stomach a hormonal message is transmitted to the parietal cell causing the pump-containing vesicles to move to the apical cell surface and begin secreting acid ◙ histamine for example binds at a cell surface receptor triggering cellular signal transduction

Steps in Cellular Respiration that lead up to the ETC:

The pathways of glycolysis and TCA are rich in proteins some termed dehydrogenases.

Myotonia

ability of muscle to maintain tone.

3. Copper atoms:

all are located within complex IV ► alternate between Cu+2 and Cu+1

dehydrogenases:

enzymes that transfer pairs of electrons from substrates to molecules such as the coenzymes NAD+ and FADH (empty wheelbarrows) ► the result of their action is that H+ and e- are transferred to carrier molecules and shuttled to the inner mitochondrial membrane

ECM

extra cellular matrix: composed of proteins like collagen and fibronectin. cell's carbohydrates can latch on to these proteins and link up this way. allowing mechanical gated channel to open and close with movement.

vasopressin:

hormone that stimulates water retention by the collecting ducts of the kidney, by way of these channels.

ataxia

inability to maintain body control (loss of muscle control)

familial

inherited from parent

Active transport depends on:

integral membrane proteins that selectively bind a particular solute and move it across the membrane against its concentration gradient in a process driven by energy requiring changes in the protein's conformation.

carrier mediated:

interaction with solute, change shape, let go. (alternating conformation)

4. Ubiquinone:

lipid-soluble molecule containing a long hydrophobic chain composed of 5-carbon isoprenoid units ► is able to accept 2 electrons and 2 protons ► remains within the lipid bi-layer and moves about freely ► not to be confused with ubiquitin...plays role in recycling proteins

Long QT syndrome:

longer heart beats due to a longer period of time of polarization of QT phase.

Ca2+ channel gene mutations:

mass body muscle contractions

2. Cytochromes:

proteins that contain heme prosthetic groups ► the iron atom of heme undergoes a RedOx reaction between Fe+3 and Fe+2 when accepting and loosing a single electron ► there are at least 5 different cytochromes that differ by protein sequence around the heme structure

membrane potential

relating to non excitable cells: i.e. skin, kidney, liver etc.

simple diffusion through the lipid bilayer

small molecules (O2, N, etc) due to concentration gradient. this type of diffusion cannot become saturated. this is passive transport. //non-mediated gradient.

Facilitated Diffusion

the movement of a molecule across a membrane by the aid of a protein that does not just form a channel but instead binds the ligand and undergoes a conformational change delivering the molecule across the membrane. ► Because they operate passively, without being coupled to energy-releasing reactions, facilitated diffusion can mediate movement in both directions.

Proteins that operate in facilitated diffusion are termed:

transport proteins. ► formerly known as carrier proteins ► permease: term applied to some transport proteins that move uncharged molecules like glucose.

how does active transport differ from facilitated diffusion?

unlike facilitated diffusion this movement of molecules is against a concentration gradient and therefore, requires an energy input ◙ Active transport drives the movement of ions in only one direction. ◙ Proteins that carry out active transport are often referred to as "pumps".

Ion Gated channels:

usually closed. Regulate the movement of molecules through them. 1. Chemically or Ligand gated 2. phosphorylation 3. voltage-gated 4. Mechanically-gated

Mutations in these ions can cause Voltage Gated channel problems:

Na, Ca, K

facilitated diffusion

carrier protein grabs ion and brings into cell due to concentration gradient. can become saturated..-

uniport:

carries a only one kind of solute across the membrane

Today there are a considerable number of inherited ion channel diseases named collectively "channelopathies"

caused by:, mutations in K+,Na+, Ca2+, and Cl- channels that are known to exist in human and animal models.

what is the charge of an animal cell's membrane potential??

-70mV

Regulation of glucose uptake by insulin-stimulated exocytosis:

-some tissues are able to presort premeases to other locations in the cell besides the membrane (i.e. fat cells and muscle cells) they are done this by being endocitized by the cell and brought into vesicles.

hormones that will retain H20

AVP, Arginne vasopressin, Antidiuretic

The four ETC complexes are composed of 5 types of membrane-bound electron carrier proteins.:

All of these, except ubiquinone, accept and donate electrons at prosthetic groups. 1. Flavoproteins: 2. Cytochromes 3. Copper atoms: 4. Ubiquinone: 5. Iron-sulfur:

active transport

moving against the gradient (needs ATP) known as protein pump/ Na ATPase. Facilitated diffusion //can become saturated.

Voltage Gated Channels fall into two categories:

multi or monomeric. driving force is concentration gradient.

Diffusion of Water:

Osmosis: movement of water readily from hypotonic solution to a hypertonic solution. Many cells are much more water permeable than explained by simple osmosis through the bi-layer.

Na channel gene mutation

myotonia issues. paralysis

hormones that will cause loss of H20

Tolyaptan, hydrochlorotyoside and conivaptan

► Congenital nephrogenic diabetes insipidus:

a mutation in this aquaporin channel, in which persons excrete large amounts of urine because their kidneys don't respond to vasopressin.

Flavoproteins:

a polypeptide bound tightly to one of two related prosthetic groups ► either Flavin Adenine Dinucleotide (FAD) or Flavin Mononucleotide (FMN) ► both groups are derived from riboflavin (vitamin B2) ► each accepts 2 protons and 2 electrons ► NADH dehydrogenase and sucinate dehydrogenase are examples

For both categories

a single (S4) transmembrane alpha helix in each subunit or in each domain serves a sensory function. As the membrane potential changes, the S4 voltage sensor shifts in such a way that the channel pore opens., ► 6 transmembrane alpha helices per domain or subuint: S1- S6 ► S4 contains positively charged amino acids necessary for opening of the channel The open pore selectively binds ions of the correct charge and size within the core of the channel pore. ► ions bind temporarily to specific charged amino acids exposed on the alpha helices in the pore These channels are either open or closed...not half-opened. However, there are two types of "closed". ► S4 can shift and close the channel, making a condition that can quickly re-open when the membrane voltage stimulus reoccurs, termed channel gating.

Example P2-type: The Sodium-Potassium Pump

a.k.a. the Na+/K+ATPase, ► found only in animals, although other species accomplish the same tasks with analogous mechanisms and proteins ► ATP phosphorylates the transporter and alter conformation from being Na+ to K+ receptive ► As the protein changes conformation, it translocates bound solutes across the membrane ► 3 Na+ ions are pumped out as 2K+ ions are pumped in per each cycle and hydrolyzed ATP

another name for transporter protein:

channel proteins or premeases

5. Iron-sulfur:

contain iron not in a heme group, but that is instead linked to inorganic sulfur atoms ► accepts and donates a single electron ► more than 12 distinct iron-sulfur centers have been identified within the mt membrane

symport:

cooperative channel which moves two substances simultaneously in the same direction, e.g. Na+ and glucose. Due to concentration gradient, Na+ moves downhill and releases enough free energy to move glucose uphill. simultaneously concentration gradient energy used

The Ca2+ concentration gradient is very large

cytosolic concentration is:, 10-7 M. ► this is 10,000 times less than that outside the cell

antiport:

exchanges one solute for another by transporting them in opposite directions - e.g. Cl- for HCO3 not simultaneously uses energy from other gradient to pull low affinity in or out acting as pump

Channel Regulation

open or gated channels.

simple diffusion through an aqueous protein-lined channel

pore proteins opens and molecules are allowed to move through the pore due to concentration gradient. this is passive transport. //non-mediated gradient. -cannot become saturated

► aquaporins:

proteins that allow the passive movement of water from one side to the other ◙ they contain a hydrophilic channel that is highly specific for water molecules ◙ are particularly prominent in cells, such as those of the kidney tubule and plant roots tetra-medic shape takes water and makes it move into or out of cell.

resting potential

relating to excitable cells: ie muscle cells and neurons

tetra-meric aquaporins:

takes water and makes it move into or out of a cell.

These glucose transporters are actually a family of proteins that transport :

the 6-C sugars. ► mammals have at least 6 different forms encoded for by separate genes, all monopeptides with 12 α-helical transmembrane segments and a polar core extending through the transporter

facilitated diffusion direction of movement depends solely on:

the diffusion gradient concentration.

glucose kinase

to keep sugars from exiting cell will phosphorylate sugar as soon as it enters the cell so sugar doesn't follow gradient anymore!

Malignant hyperthermia susceptibility:

Mass body convulsions. anesthetics lead to mass muscle contractions and elevated body temperature (deadly).

Diffusion of Ions:

Most biological membranes are impermeable to charged substances, including small ions such as Na+, K+, Ca2+, and Cl-. A variety of proteins termed ion channels have been identified. ► most ion channels are highly selective and allow only one type of ions to pass through ► the diffusion of ions through a channel is always downhill, from high to low concentration ► ion channels are bidirectional...they allow passage of ions in either direction with net flux of the ions depending on the electrochemical gradient To date, 75 different channels have been discovered. ► all are glycoproteins ► all have several α-helical membrane-spanning regions ► hydrophilic portions protrude into the aqueous cytosol or extracellular matrix Most of the ion channels that have been identified can exist in either an open or a closed confirmation. ► such channels are said to be gated ► opening and closing is regulated by physiological regulation and can be induced by a variety of factors depending on the particular channel ► therefore, all these proteins function allosterically

Gating:

Most ion channels exist in one of three possible configurations, which is regulated by various stimuli. ► closed ► open ► inactivated

Interesting Application: Some facilitated glucose transporters are regulated by hormones...like insulin.

► when secreted into the blood stream during high glucose levels, insulin stimulates MUSCLE AND FAT cells to place more glucose transporters in the plasma membrane. ◙ the # of transporters in most other parts of the body (LIKE LIVER & BRAIN) remain at a CONSTANT level as the transprter concentration in such tissues is not affected by hormone level ► a common form of Diabetes involves mutations that alter the sequences of the transmembrane segments of the transporter preventing transport across the plasma membrane **mutations in permeases ◙ glucose builds up in the blood stream ► the transporter has been sequenced and expressed in the lab ◙ when placed in an artificial membrane it carries out the same function as in the cell

► 3 important molecules formed during TCA:

◙ 2ATP ◙ 6 NADH (full wheelbarrow, formed by reducing NAD+) ◙ 2 FADH2 (full wheelbarrow, formed by reducing FADH) ► Note that 2 CO2 are formed during TCA...(what we breath out during "respiration")

Such endergonic movement of ions (in active transport) requires being coupled to an exergonic reaction such as:

◙ ATP hydrolysis ◙ absorbance of light ◙ transport of electrons ◙ flow of another substance down its concentration gradient

Mechanically-gated

◙ can sense movement of the cell membrane. Usually by cytoskeletal filaments attached to the channel protein (stretching to open)

Voltage-gated

◙ electrical state of cell require a deviation in the transmembrane potential

► 3 important molecules generated during theGlycolysis pathway:

◙ net gain of 2 ATP ◙ 2 pyruvate ◙ 2 NADH (full wheelbarrow, formed by oxidizing NAD+) that must be imported into the mitochondrial matrix if taking part in the ETC

Chemically or Ligand gated

◙ respond to the binding of specific neurotransmitter molecules or drugs. ligand is located in cells cytoplasm.

► multimeric gated channels

◙ such as the potassium channel ◙ several protein subunits combine to form the 3-D structure of the transport protein

► monomeric gated channels

◙ such as the sodium channel ◙ one large polypeptide folds into the transport protein having multiple domains.