Lectures 20-21 Membranes 3-4: Membrane Transportcbc.chem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC20... · Thermodynamics of Transport Processes ... • Sum of chemical

BIOC 460, Spring 2008

LEC 20-21: Membranes 3-4, MembraneTransport (corrected slides 37-38, p. 19,3-6-08) 1

Lectures 20-21Membranes 3-4:

Membrane TransportReading: Berg, Tymoczko & Stryer, Chapter 13, pp. 351-376Problems in textbook: chapter 13, pp. 379-380: #1, 3, 14a, 20Jmol structure of valinomycin:http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/valinomycin/vali.htmJmol structure of gramicidin:http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/gramicidin/gram1.htmAnimations of valinomycin (mobile carrier) and gramicidin (small molecule channel-former):http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC20-21_MembraneTransport/MembraneCarriersPores.html

Key Concepts• Free energy of transporting material across membrane depends on

concentration gradient across membrane:• For uncharged solutes,

• Solutes move spontaneously (ΔGt < 0) from compartment of higher concentration to compartment of lower concentration. Equilibrium: ΔG = 0 when C1 = C2

• Charged solutes: presence of a membrane potential as well as the chemical concentration gradient influences the distribution of ions:

Passive transport: spontaneous passage of solute "down" concentration and/or electrical potential gradient -- no input of free energy required

• Simple diffusion (no assistance)• Facilitated diffusion (rate enhanced by carrier or channel, generally an integral membrane protein (transporter or permease)

–rapid diffusion, "down" a concentration gradient–saturable (max. velocity depends on transporter concentration)–specific (depends on interaction of solute with transporter)–Example: GLUT1 glucose transporter in erythrocytes



Key Concepts, continuedPassive transport, continued:• GLUT1 transporter of erythrocytes

– Example of characteristics of a transporter proteins:• works by conformational changes linked to ligand binding• Rapid transport “down” concentration gradient• Saturable (shows a maximum velocity; can measure Kt analogous

to Km)• Specific

• Gated ion channels (ligand-gated or voltage-gated)– VERY rapid, ~107-108 ions/sec, "down" a concentration gradient– not saturable– degree of specificity/ion selectivity varies– Examples:

• Acetylcholine receptor of motor neurons• bacterial potassium channel• Eukaryotic sodium, potassium, and calcium channels

Terminology applying to all transporter proteins, passive or active:• Uniport (system in which one solute transported)• Cotransport (system in which transport of one solute is coupled to transport of another solute)

– Symport (different solutes transported in same direction)– Antiport (different solutes transported in opposite directions)

Key Concepts, continuedActive transport (transport of solute against its concentration gradient)– requires an exergonic process to drive the “uphill” transport• Primary active transport (transport of solute against its concentration

gradient, coupled directly to an exergonic chemical reaction, e.g.,ATP hydrolysis)

– Examples:• P-type ATPases

– Ca2+ ATPase of muscle cell sarcoplasmic reticulum– Na+-K+ ATPase of animal cell plasma membranes

• ABC transporters• Secondary active transport (transport/"flow" of one solute "down" its

concentration gradient is used to drive transport of a different soluteagainst its concentration gradient energy

– Concentration gradient of solute that “drives” the unfavorable processcomes from ATP hydrolysis

– Examples• E. coli lactose permease (H+-lactose symporter)• Na+-glucose symporter (in some animal cells

• Mechanisms of transport processes involving membrane proteinsusually involve protein conformational changes.



Learning Objectives• Terminology: membrane potential, passive transport, simple diffusion,

facilitated diffusion, ionophore, gated channel, P-type ATPase, activetransport (primary, and secondary), cotransport, symport, antiport,uniport

• What defines equilibrium for a transport process involving an unchargedsolute? (Express it in words, and in terms of concentrations at the"origin" and at the "destination", C1 and C2, and also in terms ofΔGtransport.)

• Explain free energy changes in biological transport reactions in terms ofwhether they are "favorable" (solute is moving in direction to go towardsequilibrium, ΔGtransport < 0) or "unfavorable" (would have to go in otherdirection to go towards equilibrium, ΔGtransport > 0).

• Given the equation for free energy of transport, be able to calculateΔGtransport for an uncharged solute (e.g., glucose), given theconcentrations on both sides of the membrane and the direction oftransport.

• Explain the 2 terms involved in calculating ΔGtransport for a charged solute,under what conditions the first term (concentration gradient term) wouldbe favorable, and under what conditions the 2nd term (electrical gradientterm) would be favorable.

• Briefly explain ionophores, and the difference between mobile carriersand channel-forming compounds, with one example of each.

Learning Objectives, continued• Explain whether ion channels mediate passive or active transport, and

use the acetylcholine receptor to explain how one ion channel cancontrol the flow of ions.

• Describe the GLUT1 erythrocyte glucose transporter with respect to– its biological function– kinetics of transport ("saturation" behavior: plots of Vo vs. [glucose]

and 1/Vo vs. 1/[glucose])– proposed mode of action.

• Name 2 P-type ATPases and briefly explain their biological functions.Which one consumes over 30% of the ATP in animal cells in order toestablish/maintain the [Na+] and [K+] concentration gradients andelectrical potential across the plasma membrane? Which oneestablishes/maintains the Ca2+ gradient involved in control of musclecontraction?

• Outline the proposed mechanism by which P-type ATPases couple ATPhydrolysis with "uphill" transport of solutes, with the sarcoplasmicreticulum Ca2+-ATPase as the example.

• Briefly explain how the E. coli lactose permease couples H+ transportand lactose transport, as an example of a membrane protein thatfunctions as a secondary active transporter, coupling "downhill" transportof one solute to "uphill" transport of another solute.



Thermodynamics of Transport Processes• equilibrium for a transport process (as for any process): conditions

under which ΔGt = 0.

uncharged solutes: free energy change for transporting an uncharged soluteacross a membrane depends only on concentration gradient across themembrane:

• For uncharged solute:

• where C1 = concentration at "origin"and C2 = concentration at "destination"• Uncharged solutes move from region of higher concentration to region of lower concentration (direction in which C1 > C2) so ΔGt = 0, when C1 = C2 (Equal concentrations defines equilibrium for a transport process involving uncharged solutes.)

Thermodynamics of Transport Processes, continuedcharged solutes• Electrical potential (charge gradient across membrane) influences

distribution of ions.• For ion of electrical charge Z:

– where F = Faraday constant [96.5 kJ/(V•mol)]– ΔV = membrane electrical potential (charge gradient across membrane),

in Volts.• ΔV (charge gradient, electrical potential gradient)

– can result from concentration differences across membrane for OTHERions than the one you're looking at

– Overall ΔGtransport is SUM of concentration gradient term and ΔV term– ΔV term can work either “with” or “against” concentration gradient

• You have to draw a picture of the transport process to decide on thesign of the ΔV term (decide whether that term favors or disfavors thedirection of transport of the particular ion)

• NOTE: there's no charge gradient term if solute is uncharged:ZFΔV = 0 if Z = 0.



Free Energy Change for Transport of Charged Solutes

• ΔGt depends on signs and relative magnitudes of concentration gradientterm and charge gradient term.

• Sum of chemical potential term + electrical potential term =electrochemical potential ( = ΔGt )

Does the concentration gradient of Cl– ion across this cell membranefavor transport of the ion into or out of the cell?Without doing a calculation, what would be the sign on the first term ofthe equation for free energy of transport of Cl– out of the cell?

Does the charge gradient across this cell membrane favor transport ofCl– ion into or out of the cell?Without doing a calculation, what would be the sign on the chargegradient term for free energy of transport of Cl– out of the cell?

Cout/Cin > 1, so transport out would have this term > 0, i.e. positive;concentration gradient (C2/C1 term) unfavorable to transport out of cell.

If you’ve drawn a picture like the one above, it’s clear that there’s more neg. charge inside cell than outside, so last term favors transport of a negative ion like Cl– out of cell (term would have a – sign).

Another example: Transport of Charged Solutes• Suppose intracellular [K+] = 157 mM and extracellular [K+] = 4 mM.• Suppose plasma membrane ΔV = 0.06 V, inside negative relative to outside.• For transport of K+ ions into the cell (298K), is chemical potential term

(concentration gradient) favorable or unfavorable? Its sign?• Is electrical potential term (ΔV term) favorable or unfavorable? Its sign?

• Chemical potential term: C1= 4 mM, C2= 157 mM, so C2/C1 > 1.• Thus sign of concentration term is positive (chemical potential unfavorable for transport in).• Electrical potential term: Inside of cell is negative relative to outside (info given, drawn on diagram), so charge gradient means transporting a + ion (K+) in would be favorable from a charge point of view.• ΔV term (2nd term in equation) must be negative (electrical potential favors transport of a + ion into cell).• Calculation is on posted sample problems PDF file.

Draw picture:



Thermodynamics of Transport Processes, Summary

1) thermodynamically favorable processes• ΔGt' < 0 (ΔGt' is negative)

– Uncharged solute: transport direction is favorable if moving fromarea of higher concentration to area of lower concentration (C1 > C2),direction that would result in equalization of concentrationdistribution

– Charged solute (ion): overall ΔGt' depends on 2 terms:• concentration term (chemical potential) favorable if moving in

direction that would give equalization of concentrationdistribution

• electrical potential term favorable if moving in direction thatwould result in equalization of charge distribution.

• "passive transport": any transport process in direction in which ΔGt' < 0for the process itself– Passive transport goes spontaneously because its ΔGt' is negative (no

free energy input required to make process proceed).– Favorable direction often referred to as "downhill" direction (ΔGt' < 0)

Thermodynamics of Transport Processes, Summary (continued)What if you need to move a solute in an unfavorable direction (an "uphill"

process)?2) thermodynamically unfavorable processes• ΔG't > 0 (ΔG't is positive) for the process itself• Moving a solute across a membrane against its concentration and/or

charge gradient, i.e.,• moving from area of lower concentration to area of higher concentration

and/or in direction that would make charge gradient even steeper• requires free energy coupling to another process with a greater

negative free energy change, so overall ΔG't will be negative:“active transport”

Kinds of tranport1) Passive transport

•solute moving in favorable direction, from higher concentration to lowerconcentration, and/or (for charged solutes) in direction that would reducecharge gradienta) simple diffusion

–solute moves freely across lipid bilayer; no membrane protein needed to assist process–lipophilic solutes, passing freely across membrane -- don't need help



Passive Transport, continuedb) facilitated diffusion

• specific protein required to "assist" solute to cross lipid bilayer• solutes pass "down" concentration or charge gradient, but high

activation energy to get them across membrane• Polar or charged solutes have to lose H2O molecules solvating them

in aqueous phase before they can cross hydrophobic lipid core ofmembrane.

• Protein transporter ("assisting" molecule) in membrane reducesactivation energy barrier to get polar solute, e.g., glucose, across

– by binding solute and transporting it across membrane, but onlyin direction dictated by concentration or charge gradient, OR

– by providing an opening (a channel) for solute to crossmembrane

• Solute moves only in direction dictated by concentration or chargegradient.

• Transporters and channels both display substrate specificity.• Some transporters and channels working/"open" all the time; others

"gated" (opening regulated by some signal that opens or closes)

Energy changes as hydrophilic solute crosses membrane

Nelson & Cox, Lehninger Principlesof Biochemistry, 4th ed., Fig. 11-28

Transporter protein reduceshigh activation energyfor hydrophilic solute tocross hydrophobic coreof lipid bilayer by

a) Forming noncovalentinteractions with soluteto replace interactionswith “lost” H2O ofhydration,

andb) Providing a hydrophilic

passageway acrossmembrane



Ionophores: Non-Protein Carriers and Pores (passive)

Mobile carrier, e.g., valinomycin

mobile carriers•example: valinomycin: •antibiotic•a cyclic depsipeptide (has some ester linkages as well as peptide bonds) with both D- and L-amino acids•specifically binds K+ ions•diffuses randomly from one side of membrane to the other, binding K+ where its concentration is higher, and releasing it where its concentration is lower•Monensin: similar compound specific for Na+ ions

compounds that work as transporters thatdissipate essential ion gradients, so workas poisons/antibiotics

Pore-formingcompound, e.g.,gramicidin

Pore-forming compounds•form pores (channels) in membrane through which ions can diffuse in or out of cell. •example: Gramicidin A •a peptide antibiotic with alternating D- and L-amino acids•forms a channel large enough for protons, Na+ and K+ ions to pass through, but is blocked by Ca2+

Protein Transporter Terminology• uniport: transport of just one kind of solute; e.g., ion channels, or GLUT1

glucose transporter (both passive), or Ca2+ ATPase (active transport) • COTRANSPORT: processes that COUPLE transport of more than one

soluteTypes of Cotransport:a) symport: cotransport process in which 2 solutes are obligatorily

transported in the same direction across membrane; e.g., E. coli lacpermease, protons and glucose both tranported into cell

b) antiport: cotransport process in which 2 solutes are obligatorilytranported at the same time in opposite directions acrossmembrane; e.g. eukaryotic Na+K+ ATPase

Berg et al.,Fig. 13-10



Examples of Protein Transporters (protein-mediated transport)• Passive Transport: Solute is moving in favorable direction, from higher

concentration to lower concentration, and/or (for charged solutes) indirection that would reduce charge gradient.

• Examples:1. gated ion channels, acetyl choline receptor2. glucose transporter, GLUT1

1. Gated eukaryotic ion channels• Background: Animal cells maintain steep gradient of Na+ and K+ ions

across their plasma membranes:[Na+]OUT >>> [Na+]IN

• [K+]IN >> [K+]OUT .• Membranes with this large ion concentration/charge gradient are in a state

of polarization – they have a difference in electrical potential across theirmembrane (ΔV ≠ 0).

• Generating and maintaining transmembrane ion concentration gradientscosts cell a LOT of energy (by active transport, Na+-K+ ATPase, below).

• Ion channels mediate passive transport: permit ions to dissipategradient, crossing membrane, flowing DOWN their concentration gradient,but only in the "open" conformation.

• specificity: highly selective for particular ions, though it may not beabsolute

Examples of Protein Transporters (protein-mediated transport)• Gated channels have 2 conformational states: open and closed

– Open <==> closed transition for channels regulated by somesignal:

• an electrical potential change (“voltage-gated channel”)or

• a ligand (“ligand-gated channel”)– Open states often spontaneously convert back to closed states, a kind

of built-in "timer" that determines duration of ion flow.

1) voltage-gated channels (electrical potential changes causeconformational change that opens channel for ions to rush in "down" theirconcentration gradient, as in propagation of nerve impulses [actionpotentials]).

– examples: eukaryotic sodium channel, potassium channel, calciumchannel

2) ligand-gated channels (chemical signal, e.g., the neurotransmitteracetylcholine, binds to channel to bring about conformational change)

– example: acetylcholine receptor, a non-specific cation channel (pp.370-373 in Berg et al.)



Acetylcholine Receptor, a ligand-gated channel

• Acetylcholine (ACh) – neurotransmitter – released from synaptic vesicles into synaptic cleft between nerve cells – binds to ACh receptors on postsynaptic cell membrane.

• ACh binding to receptor triggers an action potential by opening ion channel portion of receptor protein so

– Na+ ions rush into cell (down their concentration gradient), and– K+ ions rush out of cell (down their concentration gradient).

Berg et al., Fig. 13-26

(Where ACh receptors arelocated)

Acetylcholine is the gate that opens the channel.

Acetylcholine Receptor, a pentameric ligand-gated channel

Berg et al., Fig. 13.27

A, structure of single subunit of ACh receptor

B, model of open form ofpentameric ACh receptorlooking down channel fromoutside cell



Acetylcholine Receptor, proposed gating mechanism

Berg et al., Fig. 13.28

Structure of pentamericACh receptor lookingdown channel fromoutside cell

M2 helices rotatewhen ACh binds

(reconstructed fromcryoelectronmicrographs)

•Proposed structural basis for opening of channel in response to acetylcholine binding:

– Helices lining channel (white in figure below) change conformation when ACh binds, rotating along their long axis to change what kind of residues are exposed to surface of channel.

• Closed conformation: large nonpolar residue(s) block channel (Leu, Ile, Phe), so ions can't pass through.• Open conformation (with ACh bound): small polar or neutral Aas face channel (Ser, Thr, Gly), so Na+ and K+ ions can pass through.

A, closed form B, open form

Glucose Transporter GLUT12. Glucose transporter (GLUT1) in erythrocyte (red blood cell) membranes:Background:• protein-mediated diffusion (transporter) that’s NOT an ion channel• facilitates (passive) transport of glucose down its concentration gradient• Other tissues have other isoforms of glucose transporters, homologous

but products of different genes, with different affinities for glucose andrates of transport, appropriate for their metabolic roles

– e.g., Neurons have GLUT3, with low Km (high affinity for glucose), so ifblood [glucose] is low, brain gets first "dibs" on scarce glucose.

• Red blood cells depend on constant supply of glucose from blood as fuel(plasma [glucose] ~ 5 mM).

• Erythrocytes "tap" glucose as energy source via glycolysis.• GLUT1 increases rate of glucose diffusion from blood across plasma

membrane into erythrocyte by a factor of 50,000.GLUT 1 example:

1. Structure2. Kinetics3. Proposed mechanism



2. Glucose Transporter GLUT1: Sequence known, 3-D structure isn’t

• E. coli lactose permease, another transporter with 12 transmembraneamphipathic α helices -->• GLUT1 structure is thought to be similar: 2 “halves” surrounding binding pocket for carbohydrate Berg et al.,

Fig. 13-11• hydrophobic faces of helices on outer side face lipid core of membrane, and hydrophilic sides line a polar pocket that's selective for glucose

• (example) 1 amphipathic α helix of GLUT1: "Helical wheel" diagram looking down helix axis from its amino terminus, so residues are going away from you "into the page" as numbers get higher.

Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig11-30b

2. GLUT1: Kinetics of glucose transport from outside to inside of cells• Glucose doesn't just "flow in" through an open channel -- GLUT1 binds

D-glucose by hydrogen bonds to polar residues lining proposed aqueouschannel. (Binding is specific; other hexoses bind much more weakly.)

• GLUT1 binds D-glucose with high affinity and transport rate showssaturation (hyperbolic kinetics as a function of external glucoseconcentration).

Kt analogous to "Km" for an enzyme-catalyzed reaction:Kt = solute concentration that gives 1/2 the maximal velocity of transport

Vo for transport vs. [extracellular glucose], [S]out

Nelson & Cox,Lehninger Principles ofBiochemistry, 4th ed., Fig. 11-31



2. GLUT1: Proposed Transport Mechanism: 2 alternating conformations, 1 open to outside and the other open to inside of cell

• GLUT1 increases rate of transport in direction of equilibrium. • ΔGt = 0 when inside and outside concentrations of glucose are equal (C2=C1)

• All known transport proteins appear to be asymmetrically situated transmembrane proteins that alternate between 2 conformational states with ligand binding sites exposed on opposite sides of membrane.

Voet, Voet & Pratt,Fundamentals of Biochemistry, 3rd ed.Fig. 10-13

• Glucose binding pocket isn't open to both sides of membrane at same time.• Conformational equilibrium: 1 conformation open to outside and

other conformation open to inside (analogous to R<==>T for Hb or ATCase)• Net direction of transport (binding/release) depends on relative

concentrations inside and out.

Active Transport• "pumps" that carry out transport of some solute(s) in a thermodynamically

unfavorable direction]• unfavorable transport coupled to a favorable process by conformational

changes

Berg et al.,Fig. 13-2

• Source of "driving force" for active transport/conformational change: potential energy (free energy "stored") in:

1. ATP: “primary active transport”• Net hydrolysis of ATP provides free energy needed• driven by changes in conformation linked to changes in ligand binding/dissociation and covalent modification/demodification• Light-driven proton pumps are also primary active transport. (No ATP needed -- light is the source of energy to drive the pump.)

2. some solute/ion gradient: “secondary active transport”• Gradient can be "tapped" when needed, so "uphill" transport of one solute is driven by cotransport with some other solute moving "down" its concentration gradient.• driven by changes in conformation linked to changes in ligand binding/dissociation

or



Active Transport, continued1. Primary Active Transport• ATPases: common “generic” name for ATP hydrolases, whole family of

enzymes -- all hydrolyze ATP• Membrane ATPases use free energy of net ATP hydrolysis to pump ions

across membranes.• Coupling mechanism for ion movement (against concentration gradient) to

ATP hydrolysis:covalent modification linked to conformational changes associatedwith changes in ligand binding affinities

• Control of intracellular ion concentrations very important physiologically, e.g.– Ca2+ (concentration regulated in cell and in intracellular organelles; Ca2+ a signal for many cellular processes)– Na+ and K+: primary active transport sets up ion gradients to drive other (secondary) active transport processes

Examples of primary active transport processes:• P-type ATPases: large family of homologous ATPases, including

– Sarcoplasmic Reticulum Ca2+ ATPase of muscle cell– Na+-K+ ATPase (Na+K+ pump) of animal cell plasma membranes– gastric H+-K+ ATPase (pumps protons into out of parietal cells into stomach to generate pH < 1 in lumen of stomach)

1. Primary Active Transport -- ion transport by ATPases• Transport ATPases catalyze 2 coupled processes: "uphill" ion transport

plus ATP hydrolysis via covalent modification/de-modification of enzyme– Phosphorylation/dephosphorylation → conformational changes

altering ligand (ion) binding affinities– All use same basic catalytic mechanism: transfer of terminal phosphoryl

group from ATP to Asp side chain on enzyme → phosphoaspartate(β-aspartyl phosphate, phosphoric-carboxylic anhydride linkage), acovalent intermediate (covalent catalysis).

– Phosphorylation of Asp triggers conformational change required for iontransport.

– Hydrolytic cleavage of phosphoryl group off Asp residue triggers anotherconformational change, also required for ion transport.

Example of “uphill” ion transport by an ATPase (primary active transport)• Sarcoplasmic Reticulum Ca2+ ATPase (SERCA ATPase, or SR ATPase) (sarcoplasmic reticulum, specialized endoplasmic reticulum in muscle cells)



Sarcoplasmic Reticulum Ca2+ ATPase (SERCA ATPase, or SR ATPase)Physiological background/context of Ca2+ ATPase:Initiation of Muscle Contraction, Control by [Ca2+](Ca2+ channels and SERCA ATPase)• Muscle contraction initiated by nerve impulse delivered to muscle, producing

electrochemical signal (action potential).• Electrochemical signal spreads over sarcolemmal membrane (plasma

membrane of individual muscle cell) and into muscle fiber through specialjunctions, to sarcoplasmic reticulum ("SR", specialized endoplasmicreticulum in muscle cells for Ca2+ storage).

• Electrochemical signal triggers release of Ca2+ ions from the SR (where[Ca2+] = ~1 mM) through opening of Ca2+ channels (ion channels, largeproteins in the SR membrane, passive diffusion).

• Cytosolic concentration of Ca2+ normally only about 10–7 M (0.1 µM), too lowto trigger contraction

• Ca2+ released by SR by opening of Ca2+ channels increases cytosolicconcentration to about 10–5 M (10 µM), triggering muscle contraction.

• Ca2+ ions in cytosol, when concentration is high (as result of nerve impulse),bind to a regulatory protein (troponin C, TnC, similar in structure to CaM;TnC is part of the troponin complex in muscle fibers).

• Ca2+-bound form of TnC changes conformation and interacts through otherproteins of troponin complex (TnT and TnI) and tropomyosin to let myosininteract with actin so muscle contraction occurs.

Sarcoplasmic Reticulum Ca2+ ATPase (SERCA ATPase, or SR ATPase)Physiological background/context of Ca2+ ATPase, continued:

Relaxation -- results from activity of the "calcium pump",SR Ca2+ ATPase, a P-type ATPase

• Contraction ends when cytosolic Ca2+ is removed (concentration is lowered again) by being pumped back into sarcoplasmic reticulum sacs by Ca2+ ATPase (ATP-driven Ca2+ pumps in SR membrane)

• Ca2+ ATPase transports 2 Ca2+ ions into SR per ATP hydrolyzed.

• [Ca2+] inside SR is ~10–3 M (bound to calsequestrin).

• ~80-90% of total protein in the sarcoplasmic reticulum is Ca2+

pump (Ca2+ ATPase)!

Garrett & Grisham, Biochemistry,3rd ed., Fig. 16-23



SERCA ATPase Structure: ATPase membrane-spanning domain of 10 αhelices, plus a 3-domain (A, P, N) cytosolic "headpiece"

Berg et al., 5thed., Fig. 13-4

Berg et al., currented., Fig. 13-3

• P domain has Asp residue that gets Phosphorylated by ATP• Active site Asp (351) in P domain indicated by arrow• N domain binds Nucleotide (ATP)• A (Actuator) domain communicates conformational changes in P & N

domains to transmembrane Ca2+ binding domain• 2 Ca2+ ions (green) bind in membrane-spanning domain.

Ca2+ ionsshown on next slide

SERCA ATPase Structure: ATPase membrane-spanning domain of 10 αhelices, plus a 3-domain (A, P, N) cytosolic "headpiece"detail of Ca2+ bindingfrom previous fig. (13-4)(bb = peptide backbone groups)


• After transfer of phosphate from ATP to Asp 351 in A domain and dissociation of ADP, conformational change disrupts Ca2+ binding sites in TM domain,• so Ca2+ dissociates.



SERCA ATPase: Proposed Mechanism(Mechanism typical of P-type ATPases in general)Enzyme interconverts between 2 different conformations, E1 and E2.1) E1 conformation (not phosphorylated) binds 2 Ca2+ ions.2) E1-(Ca2+)2 binds ATP on cytosolic side of membrane and cytosolic domains

rearrange, trapping the two bound Ca2+ ions in transmembrane domain.3) Phosphoryl group transfer: Asp side chain carboxyl (nucleophile) of E1-

(Ca2+)2 attacks terminal phosphate of bound ATP, becomingphosphorylated to E1-P-(Ca2+)2 .

4) ADP dissociates from E1-P, triggering conformational change to E2-P("eversion") because in absence of bound ADP, the E1-E2 conformationalequilibrium favors E2 state. E2-P conformation has lower binding affinity forCa2+ ions (membrane domain's Ca2+-binding site disrupted).In E2 conformation, cytosolic "entry" for Ca2+ binding is "closed" (no longeraccessible for Ca2+ dissociation).

E2 state has "escape route" open for Ca2+ dissociation on other side ofmembrane into the SR lumen, so Ca2+ ions go into SR lumen.

Ion transport has been achieved. ATP has been cleaved but no hydolysis has occurred yet.5) When Ca2+ ions dissociate, phosphate is hydrolyzed off Asp residue to

release Pi (E2-P → E2).6) E2 without covalently attached phosphate "everts" again, back to E1 state.

SERCA ATPase: Proposed Mechanism



Na+-K+ ATPase (Na+-K+ pump in animal cell plasma membranes)• Animal cells: high intracellular [K+] and low intracellular [Na+] (relative to

extracellular medium)• Opposite gradients of Na+ and K+ require free energy input to maintain.• energy provided by ATP hydrolysis, catalyzed by a specific P-type ATPase:

plasma membrane Na+-K+ ATPase ("Na+-K+ pump")• couples export of 3 Na+ ions to import of 2 K+ ions, and hydrolyzes 1

ATP to ADP + Pi• coupling by conformational changes, same mechanism as Ca2+ ATPase:• E1 state binds Na+ tightly and K+ weakly.• E2 binds Na+ weakly and K+ tightly.• Reaction cycle:

– E1 binds 3 Na+ and then ATP inside cell, trapping Na+ in binding sites– E1's active site Asp phosphorylated by ATP to give E1-P-(Na+)3– ADP dissociates, triggering conformational switch: E1-P-(Na+)3 switches

conformations ("everts") to E2-P-(Na+)3– E2-P releases its 3 Na+ on outside of cells, but then binds 2 K+ from

outside cell (extracellular medium).– E2-P-(K+)2 is dephosphorylated (hydrolysis) to release Pi (E2-P-(K+)2 →

E2-(K+)2), and E2 reverts ("everts") to E1 state.– E1-(K+)2 state dissociates the 2 K+ ions on inside of cell, and binds 3

more Na+ ions, ready to do another cycle.

Importance of Eukaryotic Plasma Membrane Na+-K+ ATPase• Na+ and K+ gradients (established by Na+-K+ ATPase) are used to drive

other cellular processes• Na+ and K+ gradients:

– control cell volume– make neurons and muscle cells electrically excitable– drive active transport of other solutes like some sugars and amino acids

• Na+ and K+ gradients so important that more than 1/3 of the ATPconsumed by resting animal is used to pump these ions!

• Digitalis (drug, a mixture of cardiotonic steroids from foxglove plant'sleaves, including the compound ouabain)– Ouabain (pronounced "wah'-bane"; from waa bayyo, Somali for "arrow

poison") a potent and specific inhibitor of Na+-K+ ATPase– inhibits DEphosphorylation of E2-P form of plasma membrane Na+-K+

ATPase– effective drug for treatment of congestive heart failure– strengthens heart muscle contractions without increasing heart rate, and

thus increases efficiency of the heart(Berg et al. p. 357 explains why/how it has this effect if you're interested)



Primary Active Transport, continuedABC Transporters: another family of ATP hydrolysis-driven transporters,

unrelated to P-type ATPases• 2 membrane-spanning domains + 2 ATP-binding domains ("ATP Binding

Cassettes", which give ABC transporters their name); Examples:– human Multidrug-resistance (MDR1) protein

• extrudes (against their concentration gradient) a variety ofhydrophobic molecules from cells that express this protein

• pumps drugs out of cells before drugs can exert their effects, forexample making tumor cells resistant to chemotherapeutic agents

– CFTR (Cystic Fibrosis Transmembrane Regulator)• Channel for Cl– ions to flow down their conc. gradient out of the cell• ATP binding and hydrolysis controls opening/closing of channel by

conformational change, but doesn’t “pump” (ΔGt already < 0)• Some prokaryotic ABC transporters confer antibiotic resistance on bacteria

expressing them, pumping the antibiotics out of the cell.• Mechanism: One conformation binds solute from one side of

membrane, changes conformation, and releases it on other side ofmembrane.

• Familiar theme though details differ from P-type ATPases (nophosphorylated intermediate for ABC transporters): conformationalchanges mediated by solute binding/release, ATP binding andhydrolysis, and ADP/Pi release result in solute transport against aconcentration gradient

Active Transport, continued2. Secondary Active Transport:• Ion gradients set up and maintained by ATP hydrolysis, e.g., Na+ and K+

gradients, require energy input (expenditure of ATP) to set up andmaintain.

• The RESULTING concentration and charge gradients representpotential energy.

• That potential energy (electrochemical potential) can be "tapped" todrive other, unfavorable transport processes.

• Proteins couple "downhill" flow of one kind of ion (e.g., letting Na+ ions flowback into cell) with "uphill" transport of another ion or solute, using energy"stored" in concentration/charge gradient (originally “paid for” by ATP) todrive another otherwise unfavorable process.

• Cotransport in secondary active transport processes:a) symport: cotransport process in which downhill flow of one species is used to drive uphill flow of another solute in the same direction across membrane; e.g., lac permease: protons flow down their conc. gradient into cell, bringing lactose into cell against its conc. gradientb) antiport: cotransport process in which downhill flow of one species is used to drive uphill flow of another solute in the opposite direction across membrane; e.g. eukaryotic Na+Ca+ antiporter: Na+ flows into cell down its conc. gradient, while Ca+ is pumped out of cell against its conc. gradient.



2. Secondary Active Transport -- examples• Lactose permease of E. coli: symporter that uses H+ gradient across E. coli membrane (generated by fuel oxidation and electron transport) to let protons flow down their concentration gradient back into the cell, bringing lactose into the cell against a concentration gradient.• conformational changes linked to H+ binding (by a specific carboxyl group) and release, and lactose binding and release


Other examples of secondary active transporters• Sodium-calcium exchanger of animal cell membranes: antiporter that

couples downhill flow of 3 Na+ into cell with uphill extrusion of 1 Ca2+ out ofcell (Na+ gradient was generated by the Na+-K+ ATPase.)

• Na+-glucose symporter (in some animal cells): uses Na+ gradient(generated by the Na+-K+ ATPase) to drive import of glucose into some cellsagainst a concentration gradient, permitting the cell to concentrate glucoseto much higher concentrations than the extracellular glucose concentration.

• Energy transduction by membrane proteins: Na+-glucose symporter(secondary active transport) is driven by Na+ gradient that was generatedby Na+-K+ ATPase (primary active transport). (See figure below.)

Berg et al., 5th ed. Fig. 13-12

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Lectures 20-21 Membranes 3-4: Membrane Transportcbc.chem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC20... · Thermodynamics of Transport Processes ... • Sum of chemical