Chapter 11- Molecular Biology of the Cell (Alberts)

Membrane Barrier

-Prevents passage of small polar molecules

-Requires the cell to: transport water soluble molecules, excrete waste products, regulate ion balance, signal transduction.

-Hydrophobic interior

-Cells use specialized transmembrane proteins to transport inorganic ions and small water-soluble organic molecules across the lipid bilayer.

-The importance of membrane transport is reflected in the large number of genes in all organisms that code for transport proteins.

Two main classes of membrane proteins:

-transporters: which have moving parts to transport specific molecules across membranes

-channels: form narrow hydrophilic pores, allowing passive transmembrane movement, primarily of small inorganic ions.

Transmembrane diffusion

Depends on size and polarity.

-given enough time, virtually any molecule will diffuse across a protein-free lipid bilayer down its concentration gradient.

-the smaller the molecule, the more soluble it is (the more hydrophobic and non polar) the more readily that it will diffuse across the lipid bilayer.

-small non polar molecules such as O2 and CO2, readily dissolve in lipid bilayers and diffuse rapidly across them.

-Small uncharged polar molecules like water or urea diffuse much more slowly.

-Lipid bilayer are highly impermeable to charged molecules, no matter how small.

Permeability Coefficients

the rate of flow of a solute across the bilayer is directly proportional to the difference in its concentration on the two sides of the membrane.

-synthetic lipid bilayers are 10^9 more permeable to water than to even such small ions as Na+ and K+.

Transport proteins

-all are multipass transmembrane proteins

-allow movement of solute without interaction with membrane lipids

-transporters: substrate specific, undergo conformational changes to transfer bound solute.

-channels: weaker, less specific, have a selectivity filter. They form aqueous pores that extend across the lipid bilayer, and when they are open they allow specific solutes to pass through them and thereby cross the membrane. Occurs at a much faster rate!

-Porins:specific channels proteins that greatly increase the permeability of these membranes to water.

Passive transport (facilitated diffusion):

"downhill"

concentration gradient ( the difference in concentration between the two sides of the membrane) drives the transport.

Electrochemical gradient

When the electrical potential and membrane potential influence the transport.

Active transport

Actively transporting certain solutes across the membrane against their electrochemical gradient. "Uphill." Needs ATP or an ion gradient.

Electrical gradient

Combines the membrane potential and the concentration gradient; the two type of gradients work together to increase the driving force on an ion across the membrane (middle) or can work against each other.

H+ gradients

The pumping of hydrogen atoms sets up the electrochemical gradient across gram-negatve bacteria membranes and across the inner mitochondrial membran of the mitochondria.

-Electron transport chain helps pump hydrogen across the membrane to be used in the synthesis of ATP.

Membrane transport

Transporters behave like enzymes.

-Transporter delivers the solute unchanged to the other side of the membrane.

-Each transporter has one or more specific binding site.

-Undergoes reversible conformational changes that alternatively expose the solute-binding site first on one side of the membrane and then on the other.

Vmax-

when a transporter is saturated ( that is, when all solute binding sites are occupied), and the rate of transport is maximal. Measures the rate at which the carrier can flip between its two conformational states.

Km (binding potential)

Is the number given to represent each transporters characteristic binding affinity for its solute during that particular reaction.

-It is equal to the concentration of solute when the transport rate is half its maximum value.

Active transport can be carried out by...

1. Coupled transporters couple the uphill transport of one solute across the membrane to the downhill transport of the other.

2. ATP driven pumps couple uphill transport to the hydrolysis of ATP.

3, Light driven pumps: found mainly in bacteria and archaea, couple uphill transport to an inout of energy from light, as with bacteriorhodopsin.

4. Electrochemical potential

5. Redox potential.

How do we know that a transport is active?

By adding ATP, changing pH, we can determine transport type.

Couple transport

Uniporter: transport of one solute in one direction.

Symporter: transport of two solutes in the same direction.

Antiporter: Transport of two solutes in the opposite direction.

The free energy released during the movement of an inorganic ion down an electrochemical gradient is used as as the driving force to pump other solutes uphill, against their electrochemical gradient. This principle can work in either direction.

Secondary active transport

-Ion driven carriers are said to mediate secondary active transport.

Pumps on different cells surfaces can be used for directed transport

Ions gradients have a crucial role in driving many essential transport processes in the cell. Ion pumps that use the energy of ATP hydrolysis establish and maintain these gradients.

3 Classes of ATP driven pumps

-P-type pumps: structurally and functionally related multiples transmembrane proteins. They phosphorylate themselves. This class includes many of the ions pumps that are responsible for setting up and maintaining gradients of Na+, k+, H+, and Ca2+ across cell membranes.

-F-type pumps: constructed from multiple different subunits. Found in the plasma membrane of bacteria, inner membrane of mitochondria, and the thykaloid membrane of chloroplasts. They are called ATP synthase because they normally work in reverse. They use H+ gradient across the membrane to drive the synthesis of ATP from ADP and phosphate.

-Structurally related to the F-type pump is the V-type pump which normal pumps H+ rather than synthesize ATP. They pump H+ into organelles like lysosomes, synaptic vesicles, and plant vacuoles to acidify the interior of these organelles.

-ABC transporters: primarily pump small molecules across cell membranes, in contrast to P-Type and the F- or V- type pumps, which exclusively transport ions.

Ca2+P-type ATPase

They contain 10 transmembrane alpha-helices, three of which line a central channel that spans the lipid bilayer. In the unphosphorylated state, two helices are disrupted and form a cavity that binds two Ca2+ ions and is accessible from the cytosolic side of the membrane and the subsequent transfer of the terminal phosphate groups of the ATP to an aspartic acid of an adjacent domain lead to a drastic rearrangement of the transmembrane helices. This rearrangment disrupts the Ca2+ binding site and releases the Ca2+ ions on the other side of the membrane, into the lumen of the SR. An essential characteristic of all P-type pumps is that the pump transiently phosphorylates itself during the pumping cycle.

Plasma Membrane P-type Na+/K+ pump establishes the Na+ gradient across the plasma membrane.

-Concentration of K+ is typically 10-30 times higher inside the cell than outside.

-The pump operates as an ATP-driven antiproton, actively pumping Na+ out of the cell against its steep electrochemical gradient and pumping K+ into the cell.

-Pump hydrolyzes ATP to pump Na+ out and K+ in, also known as a Na+/K+ ATPase.

-Like an enzyme, the Na+/K+ pump can be driven in reverse, in this case to produce ATP. When the Na+ and K+ gradients are experimentally increases to such an extent that the energy stored in their electrochemical gradients is greater than the chemical energy of ATP hydrolysis, these ions move down their electrochemical gradients and ATP is synthesized from ADP and phosphate by the Na+/K+ pump.

-It has an electrogenic effect, meaning that is drives a new current across the membrane, tending to create an electrical potential, with the cell's inside being negative relative to the outside.

-The Na+/K+ pump does not have a direct role in controlling the solute concentration inside the cell and thereby helps regulate osmolarity of the cytosol. All cells contain specialized water channel proteins called aquaporins, in their plasma membrane to facilitate water flow across this membrane. Thus, water moves into or out of the cells, down its concentration gradient, a process called osmosis.

Donnan Effect

Macromolecules do not affect osmolarity, but require counter ions that do.

--> Counter ions make for effectively greater internal concentrations.

-Donnan effect leads to the cell walls wanting to swell.

ABC transporters

They constitute the largest family of membrane transport proteins and are of great clinical importance. These structural changes in the cytosolic domains are thought to be transmitted to the transmembrane segments, driving cycles of conformational changes that alternately expose substrate-binding sites on one side of the membrane and then on the other. In this way, ABC transporters use ATP binding and hydrolysis to transport small molecules across the bilayer.

-Most are unidirectional.

-In eukaryotes, they export from the cytosol. Either to an extracellular place, or a membrane bound intracellular compartment.

-This superfamily transports inorganic ions, amino acids, mono- and polysaccharides, peptides and even proteins.

Where as bacterial ABC transporters are used for import and export, but those in eukaryotes are specialized for export.

Gram negative bacteria face special challenges.

Bacteria with double membranes are called Gram-negative because they do not retain the dark blue used in gram staining.

- A multi drug resistance protein (MDR) acts by pumping hydrophobic drugs out of the cytosol. The overexertion of which in human cancer cells can make the cells simultaneously resistant to a variety of chemically unrelated cytotoxic drugs that are widely used in cancer chemotherapy. Treatment with any one of these drugs can result in the selective survival and overgrowth of those cancer cells that express more of the MDR transporter.

Channels as gates

The main types of stimuli that are known to cause ion channels to open are a change in the voltage across the membrane (voltage-gated channels), a mechanical stress (mechanically gated channels, or the binding of a ligand (ligand gated channels).

-The ligand can be either an extracellular mediator- specifically, a neurotransmitter (transmitter-gated channels)- or an intracellular mediator such as an ion (ion gated channels) or a nucleotide (nucleotide-gated channels).

-Protein phosphorylation and de-phosphorylation regulates the activity of many ion gated channels (covalent modification).

-The most common ion channels are those that are permeable mainly to K+.

-These channels are found in the plasma membrane of almost all cells.

-An important subset of channels opens even in an unstimulated or "resting" cell, and hence these channels are sometimes called K+ leak channels. They work by making the plasma membrane much more permeable to K+ than to other ions, they have a crucial role in maintaining the membrane potential across all plasma membranes.

Channel Proteins

form hydrophilic pores across membranes.

-one class of channel proteins found in virtually all animals forms gap junctions between two adjacent cells.

-It could be disastrous is channel proteins like gap junctions and porins were directly connected to the inside of a cell to an extracellular space.

-Referred to as ion channels.

-Highly selective.

-Ion channels have an advantage over carriers, in that up to 100 million ions can pass though one open channel each second, a rate that is 10^5 times greater than the fastest rate of transport mediated by any known carrier protein.

-Ion selectivity: permitting some inorganic ions to pass, but not others.

K+ channels Selectivity Channels

The puzzle was solved when the structure of a bacterial K+ channel was determined by x-ray crystallography. The channel is made from four identical transmembrane subunits, which together form a central pore through the membrane. Negatively charged amino acids concentrated at the cytosolic entrance to the pore are thought to attract cations and repel anions, making the channel cation-selective. Each subunit contributes two transmembrane crhelices, which are tilted outward in the membrane and together form a cone, with its wide end facing the outside of the cell where K+ ions exit from the channel. The polypeptide chain that connects the two transmembrane helices forms a short a helix (the pore helix) and a crucial loop that protrudes into the wide section of the cone to form the selectivity filter. The selectivity loops from the four subunits form a short, rigid, narrow pore, which is lined by the carbonyl

oxygen atoms of their polypeptide backbones.Becausethe selectivity loops of all known K+ channels have similar amino acid sequences, it is likely that they form a closely similar structure.The crystal structure shows two K+ions in single file within the selectivity filter, separated by about 0.8 nm.

The structure of the selectivity filter explains the ion selectivity of the channel. A K* ion must lose almost all of its bound water molecules to enter the filter, where it interacts instead with the carbonyl oxygenslining the filter; the oxygens are rigidly spaced at the exact distance to accommodate a K+ ion.

Gating seems to involve movement of the helices in the membrane so that they either obstruct (in the closed state) or free (in the open state) the path for ion movement. Depending on the particular type of channel, helices are thought to tilt, rotate, or bend during gating.The structure of a closed K* channel sho

Membrane Potenital

-Arises when where is a difference in the electrical charge on the two sides of a membrane, due to slight excess of positive ions over negative ones on one side and a slight deficit on the other. Such charge differences can result both from active electrogenic pumping and form passive ion diffusion.

-The Na+/K+ pumps helps maintain an osmotic balance across the animal cell membrane by keeping the intracellular concentration of Na+ low. Because there is little Na+ in the cell, other cations have to be plentiful there to balance the charge carries by the cells fixed anions- which are confined inside the cell. This balancing role is performed largely by K+, which is actively pumped into the cell by the Na+/K+ pump and can also move freely in or out though the K+ leak channels in the plasma membrane. Because of the presence of these channels, K+ comes almost to equilibrium, where an electrical force exerted by an excess of negative charges attracting K+ into the cell balances the tendency of K+ to leak out down its concentration gradient.

Nernst equation: quantifies the equilibrium condition and makes it possible to calculate the theoretical resting membrane potential if we known the ratio of internal and external ion concentrations.

V=(RT/zF)ln(Co/Ci)

V= the equilibrium potential in volts.

C0, Ci= outside and inside concentrations of the ion, respectively.

R=gas constant

T= absolutely temperature

F= Faradays constant

z=Valence charge of ion

Aquaporins

-Especially abundant in cells that must transport water at particularly high rates.

-The understand why these channels are also impermeable to H+, recall that most protons are present in cells as H3O+, which diffuses through water extremely rapidly, using a molecular relay mechanism that requires the making and breaking of hydrogen bonds, between adjacent water molecules.

-Aquaporins contain two strategically places asparagines which bind to the oxygen atom of the central water molecule in the line of water molecules traversing the pore. Because both valences of this oxygen are unavailable for hydrogen bonding, the central water molecule cannot participate in an H+ relay, and the pore is therefore impermeable to protons.

Voltage gated Cation Channels

-responsible for generating the action potentials. An action potential is triggered by a depolarization of the plasma membrane- that is by a shift in the membrane potential to a less negative value inside.

-In nerve and skeletal muscle cells, a stimulus that causes sufficient depolarization promptly opens the voltage-gated Na+ channels, allowing a small amount of Na+ to enter the cell down its electrochemical gradient. The influx of positive charge depolarizes the membrane further, thereby opening more Na+ channels, which admit more Na+ ions, causing still further depolarization. The self-amplification process, an example of positive feedback , continues until, within a fraction of a millisecond, the electrical potential in the local region of membrane has shifted from its resting potential of about -70 mV to almost as far as the Na+ equilibrium potential of about +50 mV.

-The Na+ channels have an automatic inactivating mechanism, which causes the channels to reclose rapidly even though the membrane is still depolarized. The Na+ channels remain in the inactivated state, unable to reopen, until after the membrane potential has returned to its initial negative value.

-The self-amplifying depolarization of the patch, however it sufficient to depolarize neighboring regions of membrane, which then go through the same cycle. In this way, the action potential sweeps like a wave from the initial sight of depolarization over the entire plasma membrane.

Synapses

Neuronal signals are transmitted from cell to cell. Transmission is indirect.

-Presynaptic cell is separated from the post-synaptic cell by a narrow synaptic cleft.

Neurotransmitters

A change of electrical potential in the presynaptic cell triggers it to release small molecules known as neurotransmitters, which are stores in membrane-enclosed synaptic vesicles.

-the neurotransmitter diffuses rapidly across the synaptic cell by binding to the transmitter-gated ion channels and opening them. After the neurotransmitter has been secreted, it is rapidly removed.

Transmitter-gated ion channels

Specialized for rapidly converting extracellular chemical signals into electrical signals at chemical synapses. They can produce local permeability changes, and therefore changes of membrane potential, that are graded according to the amount of neurotransmitter released at the synapse and how long it persists there.

Excitatory neurotransmitters

Open cation channels, causing an influx of Na+ that depolarizes the post synaptic membrane toward the threshold potential for firing an action potential.

Examples: acetylcholine, glutamate, and serotonin

Inhibitory Neurotransmitters

Open either Cl- channels or K+ channels and this suppresses firing by making it harder for excitatory influences to depolarize to post synaptic membrane. Many transmitters can be either excitatory or inhibitory, depending on where they are released, what receptors they bind to, and the ionic conditions that they encounter.

Examples: Gamma- amino butyric acid (GABA) and glycine

Neuromuscular Transmission

1. The process is initiated when the nerve impulse reaches the nerve terminal and depolarizes the plasma membrane of the terminal. The depolarization transiently opens voltage-gated Ca2+ channels in this membrane. As the Ca2+ concentration outside cells is more than 1000 times greater than the free Ca2+ concentration inside, Ca2+ flows into the nerve terminal. The increase in Ca2+ concentration in the cytosol of the nerve terminal triggers the local release of acetylcholine into the synaptic cleft.

2. The released acetylcholine binds to acetylcholine receptors in the muscle cell plasma membrane, transiently opening the cation channels associated with them. The resulting influx of Na+ causes a local membrane depolarization.

3. The local depolarization of the muscle cell plasma membrane opens volt- age-gated Na+ channels in this membrane, allowing more Na+ to enter,

which further depolarizes the membrane. This, in turn, opens neighboring voltage-gates Na+ channels and results in a self-propagating depolarization

(an action potential) that spreads to involve the entire plasma membrane (seeFiguref1-30).

4. The generalized depolarization of the muscle cell plasma membrane

activates voltage-gated Ca2+ channels in specialized regions of this membrane.

5. This, in turn, causes Ca2+-gated Ca2+ release channels in an adjacent region of the sarcoplasmic reticulum (SR) membrane to open transiently and release the Ca2+ stored in the SR into the cytosol. The T-tubule and SR membranes are closely apposed with the two types of channels joined together in a specialized structure. It is the sudden increase in the cytosolic Ca2+ concentration that causes the myofibrils in the muscle cell to contract.