Transporte Activo I
-
Upload
yahel-mmaya -
Category
Documents
-
view
30 -
download
0
description
Transcript of Transporte Activo I
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 1/56
11-Oct-07 J. Arreola 1
Transporte Activo de Solutos
Clase 5
Biología y Fisiología Celular
BIM-PCBBAgosto-Diciembre 2007
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 2/56
11-Oct-07 J. Arreola 2
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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 3/56
11-Oct-07 J. Arreola %
Carrier proteins cycle between conformations
conformationcange conformationcange
Carrier-me!iate! solute trans"ort
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 4/56
11-Oct-07 J. Arreola &
Carrier proteins cycle between conformations
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$whereas a single channel opening event a!allow flu' of an! thousands of ions.
Carriers e'hibit Michaelis-Menten kinetics.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 5/56
October$ 1%$ 200( J. Arreola )
Transport ediated b! *arriers
A+ ,acilitated iffusion or niport
/+ *otransport or S!port
*+ *ountertransport or Antiport
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 6/56
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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 7/56
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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 8/56
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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 9/56
October$ 1%$ 200( J. Arreola 5
#D$substrateanalog
%actose Permease PDB &P'7
The lactose permease has been crystallized withthiodigalactoside (TDG),an analog of the
substrate lactose, adisaccharide.
The substrate analog isbound in a cavity
between 2 domains, eachconsisting of transmembrane α!helices.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 10/56
October$ 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$substrateanalog
%actose Permease PDB &P'7
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 11/56
October$ 1%$ 200( J. Arreola11
Antiport (e"change diffusion)carriers e"change one solutefor another across amembrane.
#sually antiporters e"hibit$ping pong$ %inetics.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 12/56
October$ 1%$ 200( J. Arreola12
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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 13/56
October$ 1%$ 200( J. Arreola1%
4'aple of an antiport carrier+$denine nucleotidetranslocase A9:AT9e'changer catal!8es 1+1e'change of A9 for AT9across the inner itochondrial
ebrane.
A#P (−
ADP )−
mitocon!rialmatri*
a!enine nucleoti!e translocase
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 14/56
October$ 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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 15/56
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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 16/56
11-Oct-07 J. Arreola 1(
$ctive transport
S1S2
ATP
ADP + Pi
Side 1 Side 2
Active
Transport
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 17/56
11-Oct-07 J. Arreola 17
AT9-dependent ion pups are grouped into classes$ based on
transport echanis$ genetic structural hoolog!.
&-class pumps &1&' $T(ases$ in itochondria are
used for AT9 s!nthesis using the ;electrocheical gradient.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 18/56
11-Oct-07 J. Arreola 1
)-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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 19/56
11-Oct-07 J. Arreola 15
(-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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 20/56
11-Oct-07 J. Arreola 20
(-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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 21/56
11-Oct-07 J. Arreola 21
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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 22/56
11-Oct-07 J. Arreola 22
>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$000
Bl!co!lated
One ebrane doain.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 23/56
11-Oct-07 J. Arreola 2%
α-Subunit of >a= :?= AT9-ase
,our isofors have been described+ α1$ 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@.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 24/56
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 :
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 25/56
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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 26/56
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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 27/56
11-Oct-07 J. Arreola 27
>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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 28/56
11-Oct-07 J. Arreola 2
>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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 29/56
11-Oct-07 J. Arreola 25
*!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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 30/56
11-Oct-07 J. Arreola %0
3ntracellular *a2= ;oeostasis
S4@*A 9
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 31/56
11-Oct-07 J. Arreola %1
S4@*A 9up
Three 3sofors+ S4@*A1$ S4@*A2 andS4@*A%.
Seletal Euscle+ S4@*A1
S4@*A1E#+ 100$000 10 transebrane doains
Karge regulator! c!toplasic doainbetween loops & and ) wiich has one AT9
binding site and phosphor!lation doainAS9
*a2= binding pocet for 2 *a ions.
S4@*A St t
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 32/56
11-Oct-07 J. Arreola %2
S4@*A Structure
The active site$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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 33/56
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$+
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 34/56
11-Oct-07 J. Arreola %&
The 4@ *a== pup is called 23C$+arco2ndoplasic 3 eticulu Ca==-$T9ase.
3n this diagra of23C$ reactioncycle$conforationalchanges alteringaccessibilit! of*a==-binding sitesto the c!tosol or 4@luen are depictedas positionalchanges.
?eep in ind that
S4@*A is a largeprotein thataintains itstransebraneorientation.
+
+-Ca00
2
2Ca00
+cytosol membrane lumen
2Ca00
+P-Ca00
2 +P-Ca00
2
ADP
Pi
A#P
i l f
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 35/56
11-Oct-07 J. Arreola %)
3eaction cycle of 23C$
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
+cytosol membrane lumen
2Ca
+5P-Ca
2 +5P-Ca
2
ADP
Pi
A#P
3eaction cycle of 23C$
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 36/56
11-Oct-07 J. Arreola %(
3eaction cycle of 23C$
/% *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
+cytosol membrane lumen
2Ca
+P-Ca
2 +P-Ca
2
ADP
Pi
A#P
Structure of S4@*A
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 37/56
11-Oct-07 J. Arreola %7
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.
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 38/56
Topolog! of S4@*A1
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 39/56
Topolog! of S4@*A1
Two structures of S4@*A1
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 40/56
Two structures of S4@*A1
TE organi8ation
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 41/56
TE organi8ation
TE @earrangeent on *a dissociation
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 42/56
TE @earrangeent on *a dissociation
The 9 oain
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 43/56
The 9 oain
*a binding sites
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 44/56
*a binding sites
The transport c!cle
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 45/56
The transport c!cle
11-Oct-07 J. Arreola &)
Scheatic odels for four the transport
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 46/56
Scheatic odels for four the transportc!cle of S4@*A1 pup
4structura de S4@*A con 2 *a2=
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 47/56
11-Oct-07 J. Arreola &7
4structura de S4@*A con 2 *a
S4@*A en estado 42
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 48/56
11-Oct-07 J. Arreola &
S4@*A en estado 42
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 49/56
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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 50/56
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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 51/56
11-Oct-07 J. Arreola )1
Eodel of S4@*A function
Transport s!stes are functionall! coupled
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 52/56
11-Oct-07 J. Arreola )2
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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 53/56
11-Oct-07 J. Arreola )%
; :? AT9ase+ 9arietal *ells
/
Gastrinromaffin!li%e
cellsis2
8ecpt
c4-
/
l!
/
/
l!
/
/
l!
Eusion
<-*lass AT9ase
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 54/56
11-Oct-07 J. Arreola )&
< *lass AT9ase
Eodels for the structure and function of the >a=:?=
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 55/56
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
7/17/2019 Transporte Activo I
http://slidepdf.com/reader/full/transporte-activo-i 56/56
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.