Transporte Activo I

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11-Oct-07 J. Arreola 1

Transporte Activo de Solutos

Clase 5

Biología y Fisiología Celular

BIM-PCBBAgosto-Diciembre 2007




Carrier proteins cycle between conformations

Carrier proteins cycle between conformations inwhich a solute binding site is accessible on one side

of the ebrane or the other.

There a! be an interediate conforation in which abound substrate is inaccessible to either a"ueousphase.

#ith carrier proteins$ there is never an open channelall the wa! through the ebrane



11-Oct-07 J. Arreola %


conformationcange conformationcange

Carrier-me!iate! solute trans"ort



11-Oct-07 J. Arreola &


The transport rate ediated b! carriers isfaster than in the absence of a catal!st$ but

slower than with channels. A carrier transports one or few solute

molecules per conforational c!cle$whereas a single channel opening event a!allow flu' of an! thousands of ions.

Carriers e'hibit Michaelis-Menten kinetics.



October$ 1%$ 200( J. Arreola )

Transport ediated b! *arriers

A+ ,acilitated iffusion or niport

/+ *otransport or S!port

*+ *ountertransport or Antiport



October$ 1%$ 200( J. Arreola(

*lasses ofcarrierproteins

Uniport facilitated diffusion carriers

ediate transport of a single solute.An e'aple is the GLUT1 glucose carrier.

The ionophore valinomycin is also a

uniport carrier.

Uniport Symport Antiport

A A B A

B



October$ 1%$ 200( J. Arreola7

Symport (cotransport) carriersbind two dissimilar solutes(substrates) & transport them

together across a membrane.Transport of the two solutes isobligatorily coupled.



October$ 1%$ 200( J. Arreola

A gradient of one substrate$ usuall! an ion$ a!drive uphill against the gradient transport of a co-substrate.

3t is soeties referred to as secondary activetransport.

4.g+ glucose-a! symport$ in plasa

ebranes of soe epithelial cells

bacterial lactose permease$ a "! s!port carrier.



October$ 1%$ 200( J. Arreola 5

#D$substrateanalog

%actose Permease PDB &P'7

The lactose permease has been crystallized withthiodigalactoside (TDG),an analog of the

substrate lactose, adisaccharide.

The substrate analog isbound in a cavity

between 2 domains, eachconsisting of transmembrane α!helices.



October$ 1%$ 200( J. Arreola 10

The substrate is accessible onl! towhat would be the c!tosolic sideof the intact ebrane.

The transebrane α-helices areassued to ad6ust their tilt toshift access of the binding site tothe other side of the ebraneduring the transport c!cle.

#D$substrateanalog

%actose Permease PDB &P'7




Antiport (e"change diffusion)carriers e"change one solutefor another across amembrane.

#sually antiporters e"hibit$ping pong$ %inetics.




A substrate binds is transported.Then another substrate binds istransported in the other direction.

Onl! e#change is catal!8ed$ not nettransport.

The carrier protein cannot undergothe conforational transition in theabsence of bound substrate.



October$ 1%$ 200( J. Arreola1%

4'aple of an antiport carrier+$denine nucleotidetranslocase A9:AT9e'changer catal!8es 1+1e'change of A9 for AT9across the inner itochondrial

ebrane.

A#P (−

ADP )−

mitocon!rialmatri*

a!enine nucleoti!e translocase



October$ 1%$ 200( J. Arreola 1&

*riteria to recogni8e a carrier

Specificit! of the binding site

Saturation of the transport rate

*opetition for binding sites *oupling of flu'es of two transported

species



11-Oct-07 J. Arreola 1)

$ctive transport

1% $ctive transport en8!es couplenet solute oveent across aebrane to AT9 h!drol!sis.

2. An active transport pup a! be auniporter or an antiporter.



11-Oct-07 J. Arreola 1(

$ctive transport

S1S2

ATP

ADP + Pi

Side 1 Side 2

Active

Transport




AT9-dependent ion pups are grouped into classes$ based on

transport echanis$ genetic structural hoolog!.

&-class pumps &1&' $T(ases$ in itochondria are

used for AT9 s!nthesis using the ;electrocheical gradient.




)-class pumps

<acuolar pups. ;=-AT9ase is used tolower p; in secretor! and storagesvesicles as well as in l!sosoes b!concentrating ;= fro the citosol.

Neither F- or V-Class pumps form stable phosphorylated intermediates.




(-class ion pumps

(-class ion pumps are a gene fail! e'hibitingse"uence hoolog!. The! include+

a!*+!-$T(ase$ in plasa ebranes of ost

anial cells is an antiport pup.3t catal!8es AT9-dependent transport of >a= outof a cell in e'change for ?= entering.

,"!* +!-$T(ase$ involved in acid secretion inthe stoach is an antiport pup.

3t catal!8es transport of ;= out of the gastricparietal cell toward the stoach luen ine'change for ?= entering the cell.




(-class pumps cont+

Ca!!-$T(ases$ in endoplasic reticulu 4@and plasa ebranes$ catal!8e AT9-dependent transport of *a== awa! fro thec!tosol$ into the 4@ luen or out of the cell.

Soe evidence indicates that these pups a!be antiporters$ transporting protons in theopposite direction.

*a==-AT9ase pups function to eep c!tosolic

*a==

low$ allowing *a==

to serve as a signal.




The reaction echanis for a (-class ion pump involves transient covalent odification of theen8!e.

P-Class Pum"s

A#P

'

' '!

'!

'

'

'

ADP

+n,yme-

+n,yme-

Pi

2.

At one stage of thereaction c!cle$phosphate istransferred fro AT9to the carbo'!l of aBlu or Asp side-chain$foring a Chighenerg!D anh!dride

linage .(.At a later stage in thereaction c!cle$ the 9i isreleased b! h!drol!sis.




>a= :?= AT9-ase

Eantains low F>a=Gi and high F?=Gi.

3nfluences *ell <olue.

3s an integral ebrane protein fored b! α and β subunits in a 1+1 ratio.

The α subunit is the catal!tic subunit. E# H100$000.

10 ebrane doains.

AT9 and ions binding sites.

β subunit associated to α. E# H&0$000 I (0$000

Bl!co!lated

One ebrane doain.



11-Oct-07 J. Arreola 2%

α-Subunit of >a= :?= AT9-ase

,our isofors have been described+ α1$ α2$ α%$and α&.

The! differ in + >a$ ? and oubain affinities.

;ighl! hoologous. Th!roid horone increases e'pression of α2.

;eart failure decreases e'pression of α2.

3n idne!$ aldosterone upregulates α1.

α1 is located in the plasa ebrane. α2 andα% are located in icrodoains of plasaebrane-S:4@.



11-Oct-07 J. Arreola 2&

The >a=:?= pup

>;2

( ) 1 0

5 7

1 2 % &

4'tracellular

*!tosol

EgAT9

*+ m- a/ and 01 m- /

* m- a/ and *2 m- /

b :



11-Oct-07 J. Arreola 2)

Transport b! >a= :?= AT9ase

>a=

>a=

>a=

AT9

A T 9

>a=

>a=

>a=

9

>a=

>a=

>a=

9

4'ternal

9

?=

?=

99

?=

?=

?=

?=

?=

?=

*!tosol

> @ i f h > A



11-Oct-07 J. Arreola 2(

>et @eaction of the >a= pup+ A@eversible Eechanis

iicieiei P ADP K Na ATP K Na

/&&2)&2) +++↔++

++++

a/

ump

3 a/3 a/

2 /2 /

4T

4D / i

ytosol5"ternal

a/

ump

3 a/3 a/

2 /2 /

4T

4D / i

ytosol5"ternal

> 9 9 ti i i tl i * ll < l




>a= 9up 9articipes irectl! in *ell <olueEaintenance

a/

ump

3 a/3 a/

2 /2 /

4T

4D / i

ytosol5"ternal

'ubain

Net gain of

Na Salt, thus

increasing

Cell Volume

> 9 i 4l t i




>a= 9up is 4lectrogenic

a/

ump

3 a/3 a/

2 /2 /

4T

4D / i

ytosol5"ternal

Net lost ofone positive

charge

generates

small

hyperpolariz ation ~10 mV

* t li * 2 ; t i




*!tosolic *a2= ;oeostasis

*a2= 4ntr!

*a2=

@eleasefroStores

a2/a2/

a2/

a2/

a2/

a2/a2/

6758

a2/

a2/

a2/

a2/

a2/

a2/

a2/

a2/a2/

a2/

a2/

a2/

a2/

a2/

9a2/:i 0

*!;-

9a2/: 0 2"*!3

-

3 t ll l * 2= ; t i



11-Oct-07 J. Arreola %0

3ntracellular *a2= ;oeostasis

S4@*A 9




S4@*A 9up

Three 3sofors+ S4@*A1$ S4@*A2 andS4@*A%.

Seletal Euscle+ S4@*A1

S4@*A1E#+ 100$000 10 transebrane doains

Karge regulator! c!toplasic doainbetween loops & and ) wiich has one AT9

binding site and phosphor!lation doainAS9

*a2= binding pocet for 2 *a ions.

S4@*A St t




S4@*A Structure

The active site$sp/01$ whichis transientl!phosphor!latedduring catal!sis$is in a c!tosolicdoain$ far fro

the *a==binding sites.

Conformationalcoupling between activesite ebranedoains is

apparent.

2 Ca

As")5&

Muscle 1+CAPDB &+3%

membrane

!omain

cytosolic

!omain

> t @ ti f th S4@*A



11-Oct-07 J. Arreola %%

>et @eaction of the S4@*A pup

iiii ERS i ERS i P ADP H Ca ATP H Ca

/

2

44

2&&22&22 +++↔++

++++

a/

ump

2

a2/

2

a2/

2 /2 /

4T

4D / i

ytosol6758

<!=> of 6758 a2/

is boundto alse?uestrin or areticulin

9a2/:6758 free 0.2 m-

Total 9a2/:6758 0*!2 m-

6758 @olume 02!1> of ellAolume.

8eleasing all 6758 a would

increase cytosolic a to about

2 to * B-CCCC.

The 4@ *a== pup is called 23C$+



11-Oct-07 J. Arreola %&

The 4@ *a== pup is called 23C$+arco2ndoplasic 3 eticulu Ca==-$T9ase.

3n this diagra of23C$ reactioncycle$conforationalchanges alteringaccessibilit! of*a==-binding sitesto the c!tosol or 4@luen are depictedas positionalchanges.

?eep in ind that

S4@*A is a largeprotein thataintains itstransebraneorientation.

+

+-Ca00

2

2Ca00

+cytosol membrane lumen

2Ca00

+P-Ca00

2 +P-Ca00

2

ADP

Pi

A#P

i l f



11-Oct-07 J. Arreola %)

3eaction cycle of 23C$

1. 2 *a== bind tightl! fro thec!tosolic side$ stabili8ing theconforation that allows AT9to react with an active siteaspartate residue.

2. 9hosphor!lation of the activesite aspartate induces aconforational change that

L shifts accessibilit! of the2 *a== binding sites froone side of the

ebrane to the other$

L lowers the affinit! of thebinding sites for *a==.

+

+-Ca

2

2Ca


2Ca

+5P-Ca

2 +5P-Ca

2

ADP

Pi

A#P




11-Oct-07 J. Arreola %(


/% *a== dissociates into the4@ luen.

4% *a== dissociation

prootesL h!drol!sis of 9i fro the

en8!e Asp

L conforational changerecover! that causes

*a== binding sites to beaccessible again frothe c!tosol.

+

+-Ca

2

2Ca


2Ca

+P-Ca

2 +P-Ca

2

ADP

Pi

A#P

Structure of S4@*A




Structure of S4@*A

The structure of uscle Ca!!-$T(ase 23C$ has been deterined undervarious conditions$ corresponding to

stages of the reaction c!cle.

This M-ra! structure shows 5 Ca!! bound between transebrane α-

helices.



Topolog! of S4@*A1



Topolog! of S4@*A1

Two structures of S4@*A1



Two structures of S4@*A1

TE organi8ation



TE organi8ation

TE @earrangeent on *a dissociation



TE @earrangeent on *a dissociation

The 9 oain



The 9 oain

*a binding sites



*a binding sites

The transport c!cle



The transport c!cle

11-Oct-07 J. Arreola &)

Scheatic odels for four the transport



Scheatic odels for four the transportc!cle of S4@*A1 pup

4structura de S4@*A con 2 *a2=



11-Oct-07 J. Arreola &7

4structura de S4@*A con 2 *a

S4@*A en estado 42



11-Oct-07 J. Arreola &

S4@*A en estado 42



11-Oct-07 J. Arreola &5

2 Ca

As")5&

Muscle 1+CAPDB &+3%

membrane

!omain

cytosolic

!omain

6-ray structures soing large conformational canges

in te cytosolic !omain of 1+CA/ accom"anying

canges in "osition 8 tilt of transmembrane α-elices9

Scheatic @epresentation



11-Oct-07 J. Arreola )0

Scheatic @epresentation

This siplified cartoonrepresents theproposed variationin accessibilit! andaffinit! of *a==-

binding sitesduring the reactionc!cle.

Onl! 2 transebraneα-helices arerepresented$ and

the c!tosolicdoain of S4@*Ais oitted.

Ca

en,yme "os"orylation

"os"atey!rolysis

1+CA Conformational Cycle

Eodel of S4@*A function




Eodel of S4@*A function

Transport s!stes are functionall! coupled




Transport s!stes are functionall! coupled

lasma -embranea7a a ump

3 a/

3 a/

a2/

6584

2/

2/

a2/

6758

umen

a2/

6584 a2/

a2/

-4

a2/

a2/

ytosol

5"ternal

;=:?= AT9ase+ 9arietal *ells



11-Oct-07 J. Arreola )%

; :? AT9ase+ 9arietal *ells

/

Gastrinromaffin!li%e

cellsis2

8ecpt

c4-

/

l!

/

/

l!

/

/

l!

Eusion

<-*lass AT9ase



11-Oct-07 J. Arreola )&

< *lass AT9ase

Eodels for the structure and function of the >a=:?=



11-Oct-07 J. Arreola ))

AT9ase in the plasa ebrane.

This 9-class pup coprises two

copies each of a sall gl!cos!lated Nsubunit and a large subunit$which perfors ion transport.;!drol!sis of one olecule ofAT9 to A9 and 9i is coupled toe'port of three >a= ions bluecircles and iport of two ?=ions dar red triangles againsttheir concentration gradients

large triangles. 3t is not nownwhether onl! one subunit$ orboth$ in a single AT9aseolecule transports ions. b 3onpuping b! the >a=:?= AT9aseinvolves a high-energ! ac!lphosphate interediate 41H9and conforational changes$siilar to transport b! theuscle *a2= AT9ase. 3n thiscase$ h!drol!sis of the 42 I 9interediate powers transport of

a second ion ?= inward. >a=ions are indicated b! bluecirclesP ?= ions$ b! redtriangles. See te't for details.FAdapted fro 9. KQuger$ 1551$Electrogenic Ion Pumps, SinauerAssociates$ p. 17.G



See also+

an aniation of such a postulated echanisb! the lab of . ;. EacKennan.

A diagra b! *. To!oshia relating observedconforational changes to the S4@*A reaction

c!cle website of the Societ! of Beneral 9h!siologists.

http://www.utoronto.ca/maclennan/rint1.htm

http://www.sgpweb.org/symposium2005.html

http://www.sgpweb.org/symposium2005.html

http://www.utoronto.ca/maclennan/rint1.htm

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Transporte Activo I