Membrane transport processes

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Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework**

Consortium leader

PETER PAZMANY CATHOLIC UNIVERSITY

Consortium members

SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER

The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund ***

PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS

UNIVERSITY

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ORGANIC AND BIOCHEMISTRY

Membrane transport processes

www.se.hu

(Szerves és biokémia )

(Membrán transzportok)

László Csanády

http://semmelweis-egyetem.hu/

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Biochemistry: Membrane transport processes

www.se.hu http://semmelweis-egyetem.hu/

Lecture objectives

At the end of the presentation the participant will be able to 1. describe the structural background of compartmentalization

in living cells

2. understand the phenomenon of osmosis

3. interpret the directionality of transport processes based on thermodynamics

4. differentiate passive from active, primary from secondary, and electrogenic from electroneutral transport

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• phospholipid bilayer

♦polar headgroups

toward aqueous phase

♦occluded hydrophobic layer

♦fluid mosaic model (lateral diffusion)

• membrane proteins

♦integral

1. Structural organization of biological membranes

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2. Membrane permeability

• Only lipid-soluble, nonpolar substances can cross the membrane by simple diffusion:

O₂, N₂, NH₃, CO₂, fats, lipid-soluble drugs

• Substances that cannot traverse the lipid bilayer:

H₂O, ions (K⁺, Na⁺, Ca²⁺, Cl^-),

water-soluble organic compounds

(carbohydrates, aminoacids, nucleotides)

These substances can be translocated only with the help

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3. Compartments

• extracellular space

• cytoplasm

• endoplasmic reticulum (ER)

• mitochondrial matrix

• other cell organelles

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4. Concentration gradients between individual compartments

extracellular (mM) cytoplasmic (mM)

Na⁺ 140 14

K⁺ 4 140

Ca²⁺ 1 10^-4

Cl^- 110 5

cytoplasmic (nM) mitochondrial (nM).

H⁺ 80 3

cytoplasmic (mM) ER (mM) .

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5. Osmosis

Solvent moves across a

semi-permeable membrane from the more dilute solution towards the more concentrated solution.

Semi-permeable: Allows the

small solvent molecules, but not the larger solute molecules to

5.1. The phenomenon of osmosis

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5.2. Osmotic pressure

The pressure (π) required to stop osmosis.

π = c_osmRT

c_osm=osmotic concentration (total molar concentration of all dissolved particles)

π is a colligative property:

it depends only on the

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5.3. Osmotic concentration and cell volume regulation c_osm(i.c.)<c_osm(e.c.)

cell

c_osm(i.c.)=c_osm(e.c.)

normal cell

c_osm(i.c.)>c_osm(e.c.)

cell

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5.4. Osmotic properties of electrolytes Electrolytes are substances which form ions when dissolved in water.

Examples:

● salts (NaCl → Na⁺ + Cl^-)

● acids (HCl → H⁺ + Cl^-)

● bases (NaOH → Na⁺ + OH^-)

Ions are hydrated in solution – this is energetically favourable (exothermic):

the enthalpy of hydration counteracts

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Weak electrolytes: partially ionize in H

₂

O

• weak acids: CH₃COOH H⁺ + CH₃COO^-

• weak bases: NH₃ + H₂O NH₄⁺ + OH^-

Strong electrolytes: completely ionize in H

₂

O

● salts: NaCl → Na⁺ + Cl^-

● strong acids: HCl → H⁺ + Cl^-

● strong bases: NaOH → Na⁺ + OH^-

Non-electrolytes: do not ionize in H

₂

O

• alcohols: ethanol, glycerol

• thiols: mercaptoethanol

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Ionic dissociation affects colligative properties by increasing the concentration of free solute particles:

Osmotic pressure: π = (i^.c)^.RT

Exercise: intracellular osmolarity is ~0.3 osmol/l. What is the concentration of an isotonic NaCl, and CaCl₂solution?

1 mol NaCl → 1 mol Na⁺ + 1 mol Cl^- 1 mol CaCl₂ → 1 mol Ca²⁺ + 2 mol Cl^-

Van't Hoff factor (i)

2 3 1 mol CH₃COOH 1 mol H⁺ + 1 mol CH₃COO^- ≈1

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6. Thermodynamics of transport processes

6.1. Free energy of substances in solution (J/mol)

(electrochemical potential, partial Gibbs potential)

G^o = standard free energy (J/mol) R = 8.31 Jmol^-1K^-1

F = 96500 C/mol

T = 310 K (37^oC) in a mammalian organism

z = number of elementary charges of solute (-1, 0, 1, 2, 3, etc.)

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A substance is at equilibrium between two compartments (compartment 1 and compartment 2), if G₁ = G₂:

6.2.1. Uncharged substances (z=0)

at equilibrium.

6.2. Equilibrium

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6.2.2. Charged substances (z≠0)

at equilibrium (Nernst equation) At body temperature:

(where the "reversal potential" V_rev is Φ₁-Φ₂ at equilibrium)

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Under resting (static) conditions:

• substances for which the membrane is permeable must be at thermodynamic equilibrium between compartments (otherwise the substance would flow spontaneously into the compartment which provides a lower free energy...)

• substances for which the membrane is not permeable can be distributed between compartments in a way

that is far from thermodynamic equilibrium

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6.2.3. Examples

Plasma membrane:

Under resting conditions permeability for K⁺ is high.

[K⁺]_e= 4 mM [K⁺]_i= 140 mM

⇒ K⁺ is near equilibrium

⇒ V_rev= -93 mV ≈ resting membrane potential (V_m≈ -90 mV)

}

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Plasma membrane:

Under resting conditions permeability for Na⁺ is low.

[Na⁺]_e= 140 mM [Na⁺]_i= 14 mM

⇒ Na⁺ is not at equilibrium, it experiences a large thermodynamic driving force

⇒ V_rev= +60 mV >> resting membr. potential (V_m≈ -90 mV)

}

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A transport process down the electrochemical gradient ("downhill", spontaneous)

Why do many substances require specialized transport proteins for passive transport?

For kinetic reasons: transport proteins can attain transport rates orders of magnitude larger than that of simple diffusion through the lipid bilayer.

7. Passive transport

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Types of passive transporters:

• Ion channels

• Aquaporins

• Uniporters

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7.1. Ion channels

Transmembrane protein pores

7.1.1. Gating: the gate opens/closes the pore

• a stochastic process, which occurs on the time scale of protein conformational changes (10²-10⁴openings-closings / sec)

• diverse cellular signals regulate the probability that the gate is open (e.g., membrane potential – voltage-gated channels, binding of extracellular ligand – ligand-gated channels,

phosphorylation, etc.)

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7.1.2. Permeation: ion flux through the open pore

• when the gate is open, the pore conducts only one (or a few) types of ions (selectivity; e.g., Na⁺-, K⁺-,

Ca²⁺-, Cl^--, cation-channels)

• ions flow passively, down their electrochemical gradient

• ion throughput rate can approach 10⁸ ion/s

• throughput rates saturate at high ionic concentrations

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7.1.3. Cellular consequences of ion channel function 7.1.3.1. Determination of membrane potential

• the activity of ion channels determines the conductivity of the membrane for currents carried by various ions (g_Na+, g_K+, g_Cl-)

• the magnitude of the currents carried by individual ions is

, where V_X is the reversal potential for ion X

• the membrane potential is at rest if the transmembrane currents sum to zero, i.e., if

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Thus, at rest

I.e., the membrane potential is a weighted average of the ionic reversal potentials (V_i), the weights given by the conductivities for the individual ions (g_i).

Because ion-flux through open channels does not significantly

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7.1.3.2. Vectorial ion transport

(e.g. "transepithelial", across an epithelial layer)

• epithelial cells can form tight monolayers in which cells are linked via tight junctions

• vectorial ion flow across such epithelia involve active

transporters in the basolateral membrane to set up transepithelial gradients, and ion channels on the apical surface to allow passive ion flow across the apical membrane

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7.2. Aquaporins

• Transmembrane protein pores selectively permeable to water

• The driving force for water flux is osmosis

(water flows passively towards the compartment with a higher osmolarity)

Osmolarity (osmol/l):

Total concentration of all dissolved particles (c_osm =Σ_ic_i)

E.g.: osmolarity of a 140 mM NaCl solution is 280 mosmol/l

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Osmotic pressure:

The hydrostatic pressure required to stop osmosis (π_osm= c_osmRT)

E.g.: the osmotic pressure of a 140 mM NaCl

solution at room temperature is 700 kPa (= 7 atm)

• The absence or presence of aquaporins fundamentally determines the physiological consequences of vectorial transport:

♦vectorial transport in the absence of aquaporins

⇒ concentration gradients without volume changes

♦vectorial transport in the presence of aquaporins

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Biophysics of vectorial salt-water transport:

a concerted operation of ion channels and

aquaporins

iThe lipid bilayer is impermeable to both ions and water.

iCl^- permeability requires the presence of Cl^- channels or transporters

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7.3. Uniporters

• Transmembrane proteins

• Catalyze facilitated diffusion: substrate flows down its electrochemical gradient

• Substrate throughput rate follows the time scale of protein

conformational changes (10²-10⁴/s)

• Throughput rate saturates at high substrate concentrations

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• Specificity: only one type of substrate transported

• Examples:

♦ Glut-1

(glucose transporter in red blood cell membrane)

♦ Glut-5

(fructose transporter in brush border membrane)

♦ CAT

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A transport process against the electrochemical gradient ("uphill", non-spontaneous)

• Primary active transport:

"Uphill" transport is directly coupled to ATP hydrolysis

• Secondary active transport:

"Uphill" transport is coupled to "downhill"

transport of another substance (coupled transport)

8. Active transport

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8.1. Primary active transport processes

Ultimately, these transporters are responsible for the establishement of all transmembrane gradients.

• P-type ATPases: Na⁺-K⁺-ATPase, Ca²⁺-ATPase

• V-type ATPases: vacuolar H⁺ pumps

• F-type ATPases: mitochondrial F₁-F_o-ATPase

• ABC ATPases: MDR, MRP, etc.

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8.1.1. Na⁺-K⁺-ATPase (Na⁺-K⁺ pump)

• Present in the plasma membrane of all animal cells

• Pumps out 3 Na⁺ ions in exchange for 2 K⁺ ions, at the expense of hydrolysis of 1 ATP

• Responsible for maintaining the Na⁺ and K⁺ gradient, and thereby the resting membrane potential

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8.1.2. Ca²⁺-ATPase (Ca²⁺ pump)

Important for maintaining low intracellular [Ca²⁺]

• SERCA (ER membrane): Pumps 2 Ca²⁺ ions from the cytosol into the ER in exchange for 2 H⁺ ions, at the expense of

hydrolysis of 1 ATP

• PMCA (plasma membrane) : Pumps out 1 Ca²⁺ ion from the cytosol in exchange for 2 H⁺ ions, at the expense of hydrolysis of 1 ATP

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8.1.3. Vacuolar H⁺ pumps

• Present in the membranes of synaptic and secretory vesicles

• They acidify the lumen of the vesicles at the expense of ATP hydrolysis

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8.1.4. F₁-F_o-ATPase

• Present in the inner mitochondrial membrane

• Physiological role: lets 3 H⁺-s flow into the matrix (downhill transport), while it synthesizes 1 ATP from 1 ADP + P

• Reversible: when the thermodynamic gradients are reversed, pumps out 3 H⁺-s from the mitochondrial matrix at the expense of hydrolysis of 1 ATP

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8.1.5. ABC ATPases

• Transport mostly lipophylic substances at the expense of ATP hydrolysis

• Transport mechanism: substrate likely approaches the protein from the membrane by lateral diffusion ⇒ flipping into the

opposing monolayer (flip-flop mechanism)

• Examples: bile acid transporters, transporters that cause multidrug resistance

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8.2. Secondary active transporters (coupled transport) 8.2.1. Na⁺/H⁺-exchanger (NHE)

• Present in the plasma membrane

• Allows 1 Na⁺ ion to enter the cytosol (downhill transport),

while it extrudes 1 H⁺ ion

(uphill transport, see 10.2.2.)

• Protects the cell against acidification

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8.2.2. Na⁺/Ca²⁺-exchanger (NCX)

• Present in the plasma membrane

• Allows 3 Na⁺ ions to enter the cytosol (downhill transport), while it extrudes 1 Ca²⁺ ion (uphill transport)

• Important for maintaining low intracellular [Ca²⁺]

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8.2.3. Na⁺-glucose cotransporter (SGLT1)

• Located in the apical membrane of intestinal epithelial cells

• Mediates absorption of glucose from the gut

• Allows 2 Na⁺ ions to enter the cytosol (downhill transport),

while it also imports 1 glucose (against concentration gradient)

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8.2.4. HCO₃^--Cl^- exchanger (AE1, Band 3)

• Located in the red blood cell (RBC) membrane and the kidney

• Important for blood CO₂ transport and renal acid secretion

• Exchanges 1 Cl^- ion for 1 HCO₃^- ion

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• Mechanism of CO₂ transport in the blood:

In RBCs the AE1 is close to its thermodynamic equilibrium – small, tissue specific environmental changes determine the direction of its operation. In peripheral tissues RBCs take up CO₂ and release HCO₃^-, in the lung they take up HCO₃^- and release CO₂ – as a result CO₂ in the blood plasma is replaced by the more soluble HCO₃^-.

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• Concentration gradients of transported substrates

• Membrane potential:

"Electrogenic" transport processes are sensitive to membrane potential

Electrogenic: transport cycle results in net charge transfer Electroneutral: does not result in net charge transfer

• ATP-, ADP-, and P- concentration:

Important for primary active transport processes See numerical exercises (10.).

9. Factors that determine the direction of active transport

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10. Numerical exercises

10.1. Calculation of membrane potential

10.1.1. Membrane conductance values of a resting neuron are (in relative units) g_K+: g_Na+: g_Cl- = 1: 0.005: 0.1.

([Na⁺]_e=140 mM, [Na⁺]_i=14 mM, [K⁺]_e=4 mM, [K⁺]_i=140 mM, [Cl^-]_e=110 mM, [Cl^-]_i=5 mM.)

What is the resting membrane potential?

The reversal potentials for the three ions are (Nernst equation):

V_K=-93 mV, V_Na=+60 mV, V_Cl=-81 mV. Hence

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10.1.2. At the peak of the action potential, due to opening of Na⁺ channels, relative conductances are altered as follows:

g_K+: g_Na+: g_Cl-= 1: 20: 0.1.

What is the membrane potential at the peak of the action potential?

Using the new conductance values as weight factors

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10.1.3. Depolarization was caused by Na⁺ influx. How much did intracellular [Na⁺] increase, if the cell is spherical with a diameter of 20 μm, and the specific capacitance of the membrane is 0.01 pF/μm² ?

From the data the cell volume is

the cell surface is

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hence the total capacitance is

The imported charge is

How many moles of Na⁺ ions does this charge correspond to?

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What is the change in concentration due to this amount of ions?

Thus, intracellular [Na⁺] increases from 14 mM to 14.0045 mM.

The activity of ion channels can alter the membrane potential without significantly affecting ionic concentrations!

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10.2. Na⁺/H⁺ exchanger:

electroneutral secondary active transport

10.2.1. Under what conditions is the transporter at equilibrium?

The catalyzed reaction: 1 H⁺_i + 1 Na⁺_e → 1 H⁺_e + 1 Na⁺_i If ΔG<0, the process goes

in the forward direction, if ΔG>0, the process goes

in the reverse direction, if ΔG=0, there is no net transport.

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The free energy change for the above reaction:

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(electroneutral ⇒ does not depend on membrane potential).

In the cell [H⁺]_e=10^-7.4 M, [H⁺]_i=10^-7.1 M, [Na⁺]_e=140 mM, [Na⁺]_i=14 mM. Thus,

Because z_H=z_Na=1,

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10.2.2. Why is this transporter necessary?

From the Nernst equation the reversal potential for H⁺ is V_rev= –18 mV. The resting membrane potential is far more negative (approximately –90 mV), therefore, the

thermodynamic driving force for H⁺ is inward directed, and would eventually cause acidification of the cell.

Note: On the other hand, the Na⁺/H⁺ exchanger would reach equilibrium only at [H⁺]_i=10^-8.4 M (i.e., upon alkalinization of the cytosol!). Therefore, the activity of the Na⁺/H⁺ exchanger is

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10.3. Na⁺/Ca²⁺ exchanger:

electrogenic secondary active transport

10.3.1. Under what conditions is the transporter at equilibrium?

The catalyzed reaction: 1 Ca²⁺_i + 3 Na⁺_e → 1 Ca²⁺_e + 3 Na⁺_i If ΔG<0, the process goes

in the reverse direction, if ΔG=0, there is no net transport.

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(electrogenic ⇒ membrane potential sensitive).

Since z_Ca=2 and z_Na=1,

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In the cell [Ca²⁺]_e=10^-3 M, [Ca²⁺]_i=10^-7 M, [Na⁺]_e=140 mM,

[Na⁺]_i=14 mM. Thus, the reversal potential of the transporter is V_rev=-60 mV. The resting membrane potential is more negative (~

–90 mV), i.e., V_m<V_rev, and ΔG<0.

Therefore, in a resting cell the transport cycle proceeds as written: the Na⁺/Ca²⁺

exchanger pumps Ca²⁺ ions out of the

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10.3.2. Why is this transporter necessary?

From the Nernst equation the reversal

potential for Ca²⁺ is V_rev= +120 mV. At the normal resting membrane potential

(~ –90 mV) Ca²⁺ experiences a large inward directed thermodynamic driving force. The cell can maintain its extremely small intracellular Ca²⁺ concentration only with the help of active mechanisms – the Na⁺/Ca²⁺ exchanger is one of these.

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10.3.3. Under certain conditions intracellular [Na⁺] can rise

significantly in a cell, and this can lead to "reversal" of the NCX, i.e. to extrusion of Na⁺ from the cell in exchange for Ca²⁺ uptake.

Calculate – assuming that the other parameters ([Ca²⁺]_e, [Ca²⁺]_i, [Na⁺]_e, and V_m) remain unchanged – the intracellular [Na⁺] at

which the transporter reverses.

Expressing [Na⁺]_i from the above equation

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10.4. Na⁺/K⁺ pump: electrogenic primary active transport 10.4.1. Under what conditions is the transporter at equilibrium?

The catalyzed reaction:

3 Na⁺_i + 2 K⁺_e + ATP_i → 3 Na⁺_e + 2 K⁺_i + ADP_i + P_i If ΔG<0, the process goes

in the reverse direction,

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Since z_Na= z_K=1,

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The standard free energy change for ATP hydrolysis is

Therefore, at body temperature,

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In the cell [Na⁺]_e=140 mM, [Na⁺]_i=14 mM, [K⁺]_e=4 mM, [K⁺]_i=140 mM, [ATP]_i=5 mM, [P]_i=5 mM, [ADP]_i=10^-3 M (check unit!!).

Thus, V_rev=-131 mV.

The resting membrane potential is more positive (~ –90 mV), i.e., V_m>V_rev, and ΔG<0. Therefore, under resting conditions the transport cycle proceeds as written: the Na⁺/K⁺ pump pumps Na⁺ ions out of the cell.

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10.5. Transport processes that involve bicarbonate (HCO₃^-):

Due to the equilibrium CO₂(aq) + H₂O ↔ H₂CO₃ ↔ HCO₃^- + H⁺, concentrations [H⁺], [HCO₃^-], and [CO_2(aq)] in solution are always related to each other through the following equilibrium equation:

10.5.1. What is the reversal potential for HCO₃^-, if pH_e=7.4, pH_i=7.1, and CO₂ can diffuse freely through the membrane?

Because and

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Substituting into the Nernst equation we get

Note, that V_rev for HCO₃^- is always identical to V_rev for H⁺!

10.5.2.The Na⁺/HCO₃^- - Cl^-/H⁺ exchanger transports 1 Na⁺ and 1 HCO₃^- ion in exchange for 1 Cl^- and 1 H⁺. What is the direction of the transport under physiological conditions? ([Na⁺]_e=140 mM, [Na⁺]_i=14 mM, [Cl^-]_e=110 mM, [Cl^-]_i=5 mM, pH_e=7.4, pH_i =7.1)

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The catalyzed reaction: 1 Na⁺_e + 1 HCO₃^-_e + 1 Cl^-_i + 1 H⁺_i →

→ 1 Na⁺_i + 1 HCO₃^-_i + 1 Cl^-_e + 1 H⁺_e

Because the process is electroneutral, the free energy change for the above reaction is:

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Because we obtain

I.e.,

Substituting the data into the left-hand side of this equation we obtain 0.55 which is smaller than 1, i.e., ΔG<0. Thus, under the specified conditions the transport cycle proceeds as written,

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11. Recommended literature

Orvosi Biokémia (Ed. Ádám Veronika): pp 421-447

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Fizikai kémia I. (Ed. P.W. Atkins): pp 152, 209-233