Ch 12 Lipids And Cell Membranes

Front 1

What is a pump? Carrier? Channel?

Back 1

Pumps use free energy to drive uphill transport of ions or molecules - active transport
Carriers mediate mediate the transport of ions and small molecules across the membrane without consumption of energy (ATP)
Channels provide a membrane pore through which ions can flow very rapidly in a thermodynamically downhill directions - passive transport (facilitated transport) - display substrate specificity by facilitating transport of some ions

Front 2

Difference between primary and secondary active transport? What is an ATP-driven pump?

Back 2

Primary active transport uses the free energy of ATP hydrolysis to drive the movement of ions against their [gradients]. Secondary active transport utilizes the gradient of one ion to drive the transport of another against its gradient

Front 3

What are 2 types of ATP-driven pumps?

Back 3

P-type ATPases (ATP synthase)
ATP-binding cassette (ABC) transporters

Front 4

What is a gap junction?

Back 4

A cell-to-cell channel that allows the flow of metabolites or ions between cells

Front 5

What does the expression of transporters show about the cell?

Back 5

The expression of a specific set of transporters in its plasma membrane defines the cell's characteristics
-determines the ionic composition inside cells and the compounds that can be taken up
-glucose metabolism: type of GLUT transporter determines type of tissue

Front 6

What are lipophilic molecules

Back 6

molecules that can pass through cell membranes
-steroid hormones

Front 7

What is simple diffusion?

Back 7

Movement of molecules down their [gradient]

Front 8

Second Law of Thermodynamics and movement of molecules

Back 8

Molecules move from a region of higher concentration to one of lower concentration

Front 9

Equation for free-energy change in transporting an uncharged species from side 1 (concentration c1) to side 2 (conc. c2)

Back 9

^G = RT ln (c2/c1)

R = .008315 kJ/mol/deg
T = 298 K usually

Front 10

Equation for free-energy change in transporting a charged species from side 1 (concentration c1) to side 2 (conc. c2)

Back 10

^G = RT ln (c2/c1) + ZF^V

where Z is the charge of the transported species, ^V is the potential in volts across the membrane, F is 96.5 kJ/V/mol

Front 11

A transport process must be ACTIVE when ^G is -
and PASSIVE when ^G is -.

Back 11

positive and negative

Front 12

Most animal cells contain a - [K+] and a - [Na+] relative to the external medium. These ionic gradients are generated by a transport system called the -.

Back 12

a high [K+] and a low [Na+]; Na+-K+ pump/ATPase

Front 13

Suppose [Na+] outside and inside are 143 and 14 mM, and [K+] are 4 and 157 mM. At a membrane potential of -50 mV and 37 C, determine the free-energy change in transporting 3 mol of Na+ out of the cell and 2 mol of K+ into the cell. Can ATP hydrolysis provide enough energy for this process?

Back 13

Na+ and K+:
^G = RT ln (c2/c1) + RT ln (c2/c1)
^G = (3)(.008315)(310) ln (143/14) + (2)(.008315)(310) ln (157/4)
^G = 3(5.99) + 2(9.46) = +36.9 kJ/mol

Front 14

About how much free energy is generated from ATP hydrolysis?

Back 14

-50 kJ/mol

Front 15

Name two evolutionary-related ion pumps

Back 15

1. sarcoplasmic reticulum Ca2+ ATPase (SERCA): transports Ca2+ out of cytoplasm and into sarc. reticulum of muscle cells
2. gastric H+-K+ ATPase: responsible for pumping protons into stomach to lower pH

Front 16

What are P-type NTPases?

Back 16

hundreds of known homologs that form a key phosphorylated intermediate - a phosphoryl group from ATP is linked to the side chain of a specific conserved asp residue in the ATPase to form phosphorylaspartate

Front 17

P-type ATPases couple - and - to pump ions across membranes

Back 17

phosphorylation and conformation changes

Front 18

P-type ATPase, which constitutes -% of protein in the -, plays an important role in -.

Back 18

80%
sarcoplasmic reticulum membrane
relaxation of contracted muscle

Front 19

- is triggered by an abrupt rise in the - calcium ion level. Subsequent muscle relaxation depends on the rapid removal of Ca2+ from the - into the -, by -.

Back 19

muscle contraction; cytoplasmic; cytoplasm;sarcoplasmic reticulum; SERCA

This pump maintains a [Ca2+] of ~0.1 uM in the cytoplasm compared with 1.5 mM in the sarc. reticulum

Front 20

Describe the binding site of SERCA (P-type ATPase)

Back 20

A transmembrane domain consists of 10 alpha helices, and includes 2 Ca2+ binding sites. Each Ca2+ is coordinated to 7 O atoms from glu, asp, thr, asn residues, and carbonyl groups and H2Os.
A large cytoplasmic headpiece constitutes nearly half the molecular weight of the protein and has 3 distinct domains

Front 21

Describe the domains of SERCA (P-type ATPase)

Back 21

N domain binds the ATP nucleotide
P domain accepts the phosphoryl group on a conserved aspartate residue
A domain serves as an actuator, linking changes in N and P domains to the transmembrane part - this domain rotates when Ca2+ is bound

Front 22

Detailed mechanism of Ca2+ pumping by SERCA (P-type ATPase)

Back 22

1. The catalytic cycle begins with enzyme in unphosphorylated state with 2 Ca2+ bound (E1-(Ca2+)2). SERCA can exchange Ca2+ but only with cytoplasmic Ca2+
2. In E1 conformation, SERCA can bind ATP. N, P, and A domains rearrange as they close around the bound ATP, but there is no substantial rearrangement in the transmembrane domain
3. The Pi is cleaved and transferred from ATP to Asp of the P domain
4. Upon ADP release (P stays on Asp), SERCA changes overall conformation, INCLUDING the transmembrane domain - E2 or E2-P when phosphorylated. Eversion has occurred. In E2-P, the Ca2+ binding sites become disrupted and calcium are released to the other side.
5. The phosphorylaspartate residue is hydrolyzed to release inorganic phosphate.
6. With the release of phosphate, the interactions stabilizing the E2 conformation are lost, and the enzyme reverts to E1.

Front 23

What is eversion?

Back 23

The process of interconverting the E1 and E2 conformations

Front 24

How is Na+-K+ ATPase similar to SERCA?

Back 24

Na+-K+ ATPase is a a2B2 tetramer - its a subunit is homologous to SERCA and includes an analogous KEY ASPARTATE RESIDUE. The B subunit doesn't directly take part in ion transport. 3 Na+ ions bind from inside of cell to E1 conformation and 2 K+ ions bind from outside the cell to the E2 conformation.

Front 25

cardiotonic steroids

Back 25

digitoxigenin and ouabain
-strong effects on the heart
These inhibit the dephosphorylation of the E2-P form of ATPase when applied on the EXTRACELLULAR face of the membrane

Front 26

digitalis

Back 26

A mixture of cardiotonic steroids derived from the dried leaf of foxglove plant, the compound increases the force of contraction of heart muscle.
1. Inhibition of Na/K pump leads to higher [Na+] inside cell.
2. A diminished Na gradient leads to an increase in INTRACELLULAR Ca2+ by the Na/Ca exchanger, which enhances cardiac muscle contraction.

Front 27

Cardiotonic steroids inhibit the - of the - form of ATPase when applied to the - face of the membrane.

Back 27

dephosphorylation; E2-P; extracellular

E2-P + H2O -x-> E2 + Pi

Front 28

What enzymes maintain membrane asymmetry?

Back 28

flippases transport lipids such as phosphatidylserine from the inner to the outer leaflet of the bilayer

Front 29

Humans have how many P-type ATPases?

Back 29

70

Front 30

multidrug resistance

Back 30

The phenomenon where the development of resistance to one drugs makes the cells less sensitive to a range of other compounds
-This was found to correlate with the activity of an ATP-dependent pump - multidrug-resistance (MDR) protein or P-glycoprotein. When cells are exposed to a drug, the MDR pumps the drug out

Front 31

MDR protein

Back 31

An ATP-dependent pump that extrudes a wide range of small molecules.
Also known as P-glycoprotein

Front 32

Structure of MDR

Back 32

4 domains:
2 membrane spanning domains
2 ATP-binding domains -> ATP-binding cassettes (ABCs) that are homologous to bacteria and archaea

Front 33

ABC transporters

Back 33

are a large family of homologous proteins composed of 2 transmembrane domains called ATP-binding cassettes (ABCs)
-members of the P-loop NTPase superfamily
-multidrug-resistance protein
-Vibrio cholerae lipid transporter: N-terminal is membrane-spanning, and C-terminal contains the ABC

Front 34

Mechanism of ABC protein transport

Back 34

1. The catalytic cycle begins with the transporter free of both ATP and substrate. The transporter can interconvert between closed and open forms
2. Substrate enters the central cavity of the open form of the transporter from inside the cell. Substrate binding induces conformational changes in the ATP-binding cassettes that increase their affinity for ATP
3. 2 ATP bind to the ABC's, changing their conformations so the 2 domains interact strongly with one another
4. The strong interaction between the ATP-binding cassettes induces a change in the relation between the 2 membrane-spanning domains, releasing the substrate to the outside of the cell.
5. The hydrolysis of ATP with water and the release of ADP and inorganic phosphate reset the transporter for another cycle.

Front 35

Difference between eukaryotic and prokaryotic ABC transporters

Back 35

Eukaryotic ABD transporters export molecules from INSIDE the cell, whereas prokaryotic ABC transporters import specific molecules from OUTSIDE the cell.

Front 36

How are ABC transporters different from P-type ATPases? How are they similar?

Back 36

They use a different mechanism to couple the ATP hydrolysis reaction for conformational changes.
The net result is the same: the transporters are converted form one conformation capable of binding substrate from one side, to another that releases the substrate on the other side.

Front 37

What are carriers?

Back 37

Carriers are proteins that transport ions/molecules across the membrane without ATP hydrolysis.
They can couple the thermodynamically unfavorable flow of one species UP a [gradient] to the favorable flow of another species DOWN a [gradient]. - SECONDARY ACTIVE TRANSPORT
-ANTIPORTERS or SYMPORTERS

Front 38

Why is lactose permease important?

Back 38

It is part of the largest family of secondary transporters with 12 transmembrane helices that appear to have arisen by duplication and fusion of a membrane protein with 6 transmembrane helices.
-lactose permease of Ecoli uses the H+ gradient across the membrane generated by fuel molecules to drive the uptake of lactose and other sugars against a concentration gradient. It has been extensively studied

Front 39

Structure of lactose permease

Back 39

2 halves, each of which comprises 6 membrane spanning alpha helices that are irregular. These halves are separated and joined by a polypeptide.

Front 40

Mechanism for lactase permease (symporter)

Back 40

1. The cycle begins with 2 halves oriented so that the opening to the binding pocket faces outside the cell. A proton binds to possibly Glu
2. In the protonated form, the permease binds lactose from outside the cell.
3. The structure everts to the form observed in the crystal structure
4. The permease releases lactose to the inside of the cell
5. The permease releases a proton to the inside of the cell.
6. The permease everts to complete the cycle

Front 41

What is a nerve impulse?

Back 41

An electrical signal produced by the flow of ions across the plasma membrane

Front 42

The interior of a neuron contains a high concentration of - and a low concentration of -. These gradients are generated by the -.

Back 42

high K+; low Na+; Na/K ATPase

Front 43

resting membrane potential

Back 43

-60 mV

Front 44

An action potential is generated when the membrane potential is

Back 44

depolarized beyond the threshold (-60 to -40 mV) and becomes positive within about a millisecond to attain +30 mV before repolarizing.

Front 45

Depolarization of the membrane beyond the threshold level leads to an increase in permeability to -. - ions begin to flow - the cell because of the large e-chemical gradient across the plasma membrane. The entry of - further depolarizes the membrane, leading to further increase in its permeability. This - feedback leads to a very rapid and large change in membrane potential. The membrane SPONTANEOUSLY becomes less permeable to - and more permeable to -. Consequently, - flows -, and membrane potential returns to - value.

Back 45

Na+; into the cell; Na+; positive feedback
Na+; K+; outward; negative

Front 46

Type of channels in neurons?

Back 46

Ion channels - capable of ion-transport that are >1000 times as fast as pumps and carriers

Front 47

Direct evidence for the existence of - channels was provided by the - technique.

Back 47

ion channels; patch-clamp

Front 48

shaker gene

Back 48

The shaker gene encodes a K channel that contains sequences corresponding to segments S1 through S6 in a repeat unit of the Na channel. Thus, a K channel subunit is homologous to a Na channel repeat.
K channel purification are much more difficult to purify due to low abundance and lack of known high-affinity ligands

Front 49

Ion specificity in the K channel

Back 49

1. The pore facing the cell interior has a diameter of about 10 A; along the way the central cavity is 8 A. The opening and central cavity are filled with water, and a K+ can fit in without losing its water shell.
~2/3rds of the way, the pore becomes more constricted to 3 A, and K+'s must give up their water molecules and interact directly. THE CHANNEL STRUCTURE REDUCES MEMBRANE THICKNESS BY ALLOWING THE SOLVATED IONS TO PENETRATE INTO THE MEMBRANE BEFORE THE IONS DIRECTLY INTERACT WITH THE CHANNEL.
2. A selectivity filter determines the preference for K+ over other ions (TVGYG) - K+ interacts with carbonyls of the peptide

Front 50

How is selectivity achieved? How is Na+ rejected from the K+ channel even if it is small enough to pass through the pore?

Back 50

The channel pays the cost of dehydrating K+ by providing compensating interactions with the carbonyl oxygen atoms lining the selectivity filter. These oxygen atoms do not interact favorably with Na+ because the ion is too small; they are rejected because they have a HIGHER COST OF DEHYDRATION. Na+ stays hydrated and cannot pass through the channel.

Front 51

How is the paradox of tight binding and rapid transfer resolved?

Back 51

MULTIPLE-BINDING SITE
The K+ channel has 4 binding sites - each subsequent ion repels the previous one, causing it to shift up the channel and push up any already-bound K+

Front 52

Voltage-gated membrane channels

Back 52

change conformation in response to changes in membrane potential

Front 53

S5 and S6

Back 53

membrane spanning regions (pore region)

Front 54

S1 through S4

Back 54

apparatus that opens the pore

Front 55

In voltage-gated K+, S1 through S4 form domains called "-" that extend from the core of the channel

Back 55

paddles
One is S4, the voltage sensor

Front 56

Model for voltage gating

Back 56

In the closed state, the paddles lie in a 'down' position. On depolarization, the cytoplasmic side of the membrane becomes for positively charged, and the paddles are pulled up. In this position, they pull the 4 sides of the base apart, increasing access to the selectivity filter and opening the channel

Front 57

Evidence that support the ball-and-chain model for channel inactivation

Back 57

-cleavage with trypsin produced channels that stayed open after depolarization
-mutant lacking N-terminal residues did not inactivate after depolarization
-lengthening chain slows inactivation; shortening chain speeds up inactivation

Front 58

ligand-gated channel

Back 58

gated by the presence of specific ligands
-acetylcholine

Front 59

acetylcholine receptor

Back 59

-pentamer (a2, B, y, del) ring
-the subunits have similar sequences; genes arose by duplication and divergence
-each subunit has a large extra cellular domain
-acetylcholine binds at the a-y and a-del interfaces
-~ 5-fold symmetry, with its 5 similar subunits
-binding causes structural alteration that initiates rotations of the a-helical rods lining the pore

Front 60

Nernst eq.

Back 60

Veq = -(RT/zF) ln ([x]in/[x]out)

Eq will be achieved when the driving force due to [gradient] is balanced by the electrostatic force resisting the motion of an additional charge

Front 61

Generation of an action potential

Back 61

1. acetylcholine is released and binds to a receptor, opening it in less than a ms. NON SPECIFIC CATION CHANNEL that causes Na+ and K+ to flow in and out, respectively. The value approaches the eq potential of the ions, -20 mV.
2. At -40 mV, the voltage-sensing paddles of Na+ channels are pulled into the membrane, opening the Na+ channels. Na+ flows into cell, MP approaches Na+ EP. Voltage-sensing paddles of K+ are also pulled in, but more slowly, and open after 1 ms.
3. Inactivation call domains plug the Na+ channels, decreasing the Na+ current. Acetylcholine receptors are also inactivated.
4. With only K+ channels open, the MP drops towards K+ EP. 5. Ball domains inactivate K+ channels and the membrane returns to its original state.

Front 62

A typical nerve cell contains 100 Na+ channels/sq um. At +20 mV, each channel conducts 10^7 ions/second. How much ions flow through each channel in 1 ms?

Back 62

10^7 ions/s = 10^10 ions/ms
(10^10)^0.5 = 10^5 ions/ms in each channel

Front 63

Mutations involved in LQTS? Drugs that prolong cardiac action potential?

Back 63

Inactivation of K+ channels, or failure to traffick the channels to the membrane.
This leads to loss in permeability of K+, which slows repolarization and delays subsequent cardiac contraction.
The hERG for a K+ channel can be blocked by a therapeutic drug

Front 64

- ions and most - (sugars, aa's, nucleotides) can flow between the interiors of cells joined by -. Proteins, nucleic acids, and polysaccharides are too large. These are important for - communication.

Back 64

inorganic; metabolites; gap junctions
intracellular

Front 65

gap junctions are made of molecules of

Back 65

connexin

Front 66

aquaporins

Back 66

water channel
rate of 10^6 molecules/s
-positively charged residues toward the center of the channel prevent the transport of protons