Membrane Transport of Membrane Transport of Small Molecules and the Small Molecules and the Electrical Properties of Electrical Properties of

MembranesMembranes

Chapter 11, Chapter 11, Molecular Biology of the CellMolecular Biology of the Cell

Jin-Chung ChenJin-Chung Chen

Lipid bilayer: to separate the inner and outer environment of the cell; require special design of carrier protein to transport the water-soluble nutrient into the cell

15-30% membrane proteins exert function of transport (special case: 2/3 of the metabolic energy consumed on transport in special cell)

The higher the hydrophobicity or non-polar molecule make ease to pass through the membrane structure

Carrier protein disorder: cystinuria-cystine accumulates in the uria formed kidney stone

Two classes of transfer protein:

(1) Carrier protein (permease, transporter, pump) : for specific molecule; usually coupled with energy source

(2) Channel protein: inorganic ions; down to its concentration gradient; fast

overall, transfer proteins create electrical (because of membrane potential) and concentration gradient, in turn, used as a driving force (electrochemical gradient) to facilitate the transport process

Electrochemical gradient combines with membrane potential as a driving force

Involved conformation changeInvolved conformation change

Both carrier and channel contain specialized transmembrane domain

Transport through channel: fast transportTransport through channel: fast transport

Passive transport: facilitated diffusion for all channel protein and part of carrier (concentration or electrochemical gradient)

Active transport: against electrochemical gradient; mediated by carrier (pump); use ATP or ion gradient as energy source

Ionophores: small hydrophobic molecules (originally formed by microorganism) in membrane to transport specific ions; not coupled to energy source (down to concentration gradient)

1. Valinomycin: potassium ion (mobile)

2. FCCP: hydrogen ion (mobile)

3. A23187: calcium and magnesium ion (mobile)

4. Gramicidin A: monovalent cation (channel former)

Model of Carrier protein: passive transport

Kinetics of simple diffusion and carrier-mediated diffusion (expressed as Vmax/Km or Bmax/Kd)

Three ways of driving active transport:

(a) coupled carrier: downhill solute drive uphill solute

(b) ATP-driven: ATP hydrolysis to drive uphill solute

(c) light-driven: light energy trigger uphill solute

bacteriorhodopsinbacteriorhodopsin

Three types of carrier-mediated transport: uniport, symport ( kidney/GI epitghelial cells ) and antiport (determined by its path direction)

I. Examples of membrane transporters1. Na+-driven carriers

2. Na+-K+ pump (Na/K-ATPase; P-type transporter)

3. Ca2+ pump (P-type transporter)

4. ABC transporters

5. Neurotransmitter transporters

Application of Na+-driven carrier proteins (use ion gradient as energy source to drive solute):

a. Na+ driven glucose into the cell (stomach brush border) (symporter)

Na+ pump that regulates the cellular pH : :

a. Na+-H+ exchanger: influx of Na+ while pump out H+ (maintain the inner pH of the cell)

b. Na+-driven Cl-HCO3- exchanger: influx Na+ and HCO3

- while pump out H+ and Cl- (maintain pH of the cell)

c. Na+-HCO3- symporter of the glial cell: electrogenic, pu

mp in one Na+ and two HCO3- help to regulate the extr

acellular pH in near-by neurons during electrical activity

Transcellular transporter: apical to basolateral transport nutrients (intestinal epithelial cells: microvilli)

P-type transporter: 1. Na+-K+ pump

1. [K+]i: [K+]o = 20 : 1 whiles [Na+]i: [Na+]o = 1: 15 across the cell membrane;

2. The concentration difference is maintained by Na+-K+ pump (sodium pump; all the animal cells)

3. The pump is an antiporter, executes active transport and also is an ATPase; its consists of a catalytic subunit (1000 a.a.) and a small glycoprotein (critical for membrane docking)

4. Most important and energy consumption protein on the membrane

5. Pump 3 Na+ out in exchange of 2 K+ in (electrogenic)

Immunocytochemical localization of the Na,K pump in choroid plexus. Choroid plexus contains epithelial cells with intensely stained microvillar and intermicrovillar plasma membranes. The basal and lateral plasma membrane surfaces are not stained. Bar = 2 µm.

Schematic diagram of Na-K pump (P-type transport ATPase: can be reversed experimentally to produce ATP

Na+-dependent phosphorylation; K+-dependent dephosphorylation

Aspartic acid

Down-regulation of Na,K pumps can be initiated by dopamine via GPCR activation of endocytosis

Na+-K+ pump regulates cell volume

1. Water move along with the solutes determines the cell volume (osmolarity; tonicity)

2. Hypotonic vs. hypertonic solution

3. The fixed anions (nucleic acid and proteins) inside the cell try to pull the water molecule; while excellular Na+ (driven by pump) and Cl- (expelled by membrane potential) ions balance the force

4. Ouabain: Na-K pump inhibitor; cell swell and burst due to break up the net Na+ outflow

Calcium homeostasis: Cytoplasmic Ca2+ is regulated coordinately by a Na+/Ca2+ antiporter in plasma membranes and by P-type Ca-ATPases in plasma membranes and endoplasmic reticulum.

P-type transporter: 2. Calcium pump (Ca2+-ATPase)

1. Eucaryotic cells: [Ca2+]i: [Ca2+]o = 10-7 M: 10-3M

2. Ca2+ pump located in the endoplasmic reticulum (SR in muscle cell consist of 90% of membrane protein)

3. Ca2+ pump of the SR brings the calcium ion into the SR for storage, upon activation release the calcium into the cytosol for Ca2+-dependent cell signaling

4. Contains 10 transmembrane -helices; two calcium ion bind to calcium binding domain cause phosphorylation and lead Ca2+ release into the SR

ABC Transporter: transport ATPase1. Largest transport ATPase family (> 50 members)

2. Contains four domains: two hydrophobic domains each with six transmembrane span (function to translocate) and two ATP-binding cassettes (ABC)

3. In procaryotes, the transporter locates in the inner membrane to carry nutrients into the cell

4. The counterpart of the eucaryotic: multidrug resistance (MDR) protein, which produce resistance to drug (i.e. chloroquine and anticancer drug resistance)

5. Cyctic fibrosis: a mutation on one ABC transporter (cyctic fibrosis transmembrane regulator [CFTR] protein) that function as a Cl- channel in the epithelial

Gram-negative bacteria

(cassettes): ABC

Uptake of monoamines [dopamine, norepinephrine, serotonin..]: Transporters

1. Released monoamines will be up-take back into presynaptic terminals (DAT, NET, SERT)

2. Transporter is a Na+ and Cl+-dependent process (ouabain [Na,K-ATPase inhibitor] and veratridine [Na channel open] block uptake process)

3. Transporter is saturable, obeys Michaelis-Menten kinetics

4. 12 transmemebrane domain: intracellular phosphorylation and extracellular glycosylation

5. Uptake is energy dependent; can be blocked by tricyclic antidepressents, cocaine, amphetamine and MPTP

Features of Monoamine Transporters

Regulation of monoamine transporters can be achieved by various protein kinases

II. Ion channels and Membrane properties

Ion channels: channels mediate inorganic ion transport1. Narrow, highly selective pores that can open and close

2. Approximately 100 million ions can pass through / second (105 times greater as compared to any carrier)

3. Can not couple to energy source to perform active transport (always passive – downhill)

4. They are gated (open and close status); prolong stimulation would desensitized or inactivated ( closed; usually through phorphorylation)

5. All animal cells contain ion channels, not limited to neuron; each neuron might have more than 10 types of channel

Ion channels open in response to special stimulus

Establish the Membrane Potential1. Active electrogenic pump (such as Na-K pump) and pass

ive ion diffusion create the difference of ionic strength across the membrane; hence the difference in electrical charge on two side of the membrane

2. Major force: Na+-K+ pump and K+ leaking channels (more positive excellularly as compared to negative intracellularly)

a. High concentration of potassium ion inside the cells (due to Na-K pump) keep the electric balance of the negative charged macromolecule

b. K+ leak out down to its concentration gradient

3. Resting membrane potential: no net flow of ions across the membrane

Number of ions that move across the membrane to set up the membrane potential actually is quite minute

Nernst Equation (and Goldmann equation)

RT Co

V = zF ln Ci

V= the equillibrium potential in volts

Co/Ci = outside and inside concentration of the ion, respectively

R= the gas constant (2 cal mol-1K-1)

T= the absolute temperature (K)

F= Faraday’s constant (2.3 x 104 cal V-1 mol-1)

z= the valence (charge) of the ion

ln= logarithm to the base e

The more permeable the membrane for a given ion, the more strongly the membrane potential tends to be driven toward the equilibrum value for that ion:

changes in a membrane’s permeability to ions can cause significant changes in the membrane potential ( resting membrane potential will be determined by most permeable ion(s) )

Structural diagram of sodium channels

3-D Structure of Potassium Channel (exhibited only 2 out of 4 transmembrane subunit

s)

Separated about 8 A

Selection of ion by selectivity filter

Sodium ion is too small for carbonyl oxygen interaction and water expel

Typical nerve cell: somatodendrites and axon

Generation of an Action Potential

1. An electrical stimulus that exceeds a certain threshold triggers an electrical activity that is (a) self-propagated and (b) sustained by automatic amplification

2. Voltage-gated cation channels are responsible for generating the action potential

3. Consequence of an action potential:

a. stimulus triggers voltage-gated Na+ channel to open

b. more Na+ channel open in neighboring area, cause depolarization (reach Na+ equilibrum potential [+50mV])

c. Na+ channel inactivated

d. voltage-gated K+ channel open (bring membrane potential to resting status; reach K+ equilibrium potential [-70mv])

Dissection of an Action Potential

Consequence of an AP:

1. Voltage-dependent Na+ channels open

2. Voltage-dependent K+ channels open

3. Voltage-dependent Na+ channel inactive and close

4. Voltage-dependent K+ channel inactive and close

N-terminal 20 a.a. function as a tethered ball to inactivate the potassium channel after activation

Function of Myelination: insulated neurons to increase the rate of propagation; formed by Schwann cells (peripheral) or oligodendrocytes (central)

Node of Ranvier: myelin sheath space; most Na+ channels concentrated here

Saltatory conduction: action potential propogate node to node (travel faster and metabolic energy is conserved)

Patch clamp technique : allow current recording from single channel

Individual volgate-gated Na+ channel open in all-or-none fashion; the aggregate curent (approached by intracellular recording) across the membrane of entire cell represent the total number of channels ( not the degree) that open at a given time

Voltage-gated cation channels (Na+, K+ or Ca2+ ) are evolutionarily and structurally related

Neurotransmission from presynaptic to postsynaptic

Re-uptake

Transmitter-gated ion channel is not sensitive to membrane potential (can not produce self-excitation). They produce local permeability changes (depend on numbers of released transmitter) until can open nearby voltage-gated cation channels to initiate an action potential of the next neuron

Cl--dependent transporter

Neurotransmitter can determine post-synaptic excitation or inhibition (channel receptor)

A. Excitatory neurotransmitter: open cation channel (Ca2+, Na+) (i.e. NMDA glutamate receptor)

B. Inhibitory neurotransmitter: open Cl- or K+ channel (i.e. GABAA receptor)

Model of nicotinic acetylcholine receptor

Consequence of neuromuscular transmission1. Nerve impulse reach nerve terminal (depolarization) to

open voltage-gated Ca2+ channels; calcium ion flow into the nerve terminal triggers ACh release

2. ACh binding to acetylcholine receptor (nACh) in muscle membrane; opens the cation channels and Na+ influx initiate local depolarization

3. Local depolarization opens more voltage-gated Na+ channels and result in action potential

4. AP activates voltage-gated Ca2+ channels in transverse [T] tubules

5. In turn, causes Ca2+ release channels in SR to open and trigger actin-myosin sliding / muscle contraction

Actual look of the synapses in a cell body

Signal summation at PSP

1. Cell body and dendrites of a single neuron are covered by numerous synapse (presynaptic terminals)

2. Excitatory postsynaptic potential (EPSP): presynaptic transmitter release evoke excitatory response

3. Inhibitory postsynaptic potential (IPSP): presynaptic transmitter release evoke inhibitory response

4. All the PSPs from the dendritic tree converge at the cell body: spatial summation

5. The incoming signals summate at a given time (translate frequency into magnitude of PSP): temporal summation

6. If net excitatory input predominate: combined PSP is a depolarization; otherwise a hyperpolarization

Temporal summation:

Each presynpatic action potential arriving at a synapse produces a small postsynaptic potential (PSP). When successive action potentials arrive at the same synapse, each PSP produced adds to the tail of the preceding one to produce a larger combined PSP.

Firing frequency of an axon determines the magnitude of combined PSP

Learning and Memory(molecular mechanism of Long-term Potentiation)

1. LTP: a short burst of repetitive firing leads to subsequent single AP in the presynaptic cells evoke a greatly enhanced response in the postsynaptic neurons

2. Major brain area: Hippocampus

3. Presynaptic glutamate release evokes postsynaptic non-NMDA receptor (AMPA) activation EPSPs and action potential (Na+ inflow) remove Mg2+ ion of the NMDA receptor NMDA activation (Ca2+ inflow) trigger cascade response responsible for LTP

a. insert more AMPA receptor in postsynaptic cell

b. enhanced presynaptic glutamate strength

a. NMDA receptor b. AMPA receptor

Essential membrane components during normal synaptic transmission (excitatory

synapse)

Cellular alteration during the formation of LTP

Most critical event in LTP formation in postsynaptic neurons: New AMPA receptor insertion (exocytosis)