P-class pumps

Active
Transport
ADP + Pi
S2
S1
ATP
Side 1
Side 2
Active transport enzymes couple net solute
movement across a membrane to ATP hydrolysis.
An active transport pump may be a uniporter or
antiporter.
ATP-dependent ion pumps are grouped into
classes based on transport mechanism, as well as
genetic & structural homology.
Examples include:
 P-class pumps
 F-class (e.g., F1Fo-ATPase) & related V-class
pumps.
ABC (ATP binding cassette) transporters, which
catalyze transmembrane movements of various
organic compounds including amphipathic lipids
and drugs (e.g. P-glycoprotein  multidrug
resistance)
P-class ion pumps are a gene family exhibiting
sequence homology. They include:
 Na+,K+-ATPase, in plasma membranes of
most animal cells is an antiport pump.
It catalyzes ATP-dependent transport of
Na+ out of a cell in exchange for K+
entering.
 (H+, K+)-ATPase, involved in acid secretion
in the stomach is an antiport pump.
It catalyzes transport of H+ out of the
gastric parietal cell (toward the stomach
lumen) in exchange for K+ entering the cell.
P-class pumps (cont):
 Ca++-ATPases, in endoplasmic reticulum (ER)
and plasma membranes, catalyze ATPdependent transport of Ca++ away from the
cytosol, into the ER lumen or out of the cell.
Some evidence indicates that these pumps
are antiporters, transporting protons in the
opposite direction.
Ca++-ATPase pumps function to keep cytosolic
Ca++ low, allowing Ca++ to serve as a signal.
The reaction mechanism
for a P-class ion pump
involves transient
covalent modification
of the enzyme.
O
Enzyme-C
OH
ATP
Pi
ADP
H2O
O
Enzyme- C
O
O
P
O-
O-
P-Class Pumps
At one stage of the reaction cycle, phosphate is
transferred from ATP to the carboxyl of a Glu or Asp
side-chain, forming a “high energy” anhydride linkage (~P).
At a later stage in the reaction cycle, the Pi is released by
hydrolysis.
The Prominent Members
in the Family of P-Type
ATPases have different
number of subunits
tissue
Ca-ATPase (SR)
muscles
subunits
a
AA
994
Na,K-ATPase
ubiquitary
ab(g)
1013/295
H,K-ATPase
stomach
ab
1033/291
General Post-Albers reaction
scheme for P-type ATPases
E~P-Ca++2
E~P-Ca++2
ADP
The ER Ca++ pump
is called SERCA:
Sarco(Endo)plasmic
Reticulum
Ca++-ATPase.
ATP
2Ca++
E-Ca++2
2Ca++
E
Pi
cytosol
membrane
In this diagram of SERCA reaction cycle,
conformational changes altering accessibility of
Ca++-binding sites to the cytosol or ER lumen are
depicted as positional changes.
ER
lumen
Reaction cycle:
1. 2 Ca++ bind
tightly from the
cytosolic side,
stabilizing the
conformation that
allows ATP to react
with an active site
aspartate residue.
E~P-Ca++2
E~P-Ca++2
ADP
ATP
2Ca++
E-Ca++2
2Ca++
E
Pi
cytosol
membrane
ER
lumen
2. Phosphorylation of the active site aspartate induces a
conformational change that
• shifts accessibility of the 2 Ca++ binding sites from
one side of the membrane to the other, &
• lowers the affinity of the binding sites for Ca++.
E~P-Ca++2
E~P-Ca++2
ADP
ATP
2Ca++
E-Ca++2
2Ca++
E
Pi
cytosol
membrane
ER
lumen
3. Ca++ dissociates into the ER lumen.
4. Ca++ dissociation promotes
• hydrolysis of Pi from the enzyme Asp
• conformational change (recovery) that causes Ca++
binding sites to be accessible again from the cytosol.
Asp351
This X-ray structure
of muscle SERCA (Ca++ATPase) shows
2
Ca++ ions (colored
magenta) bound between
transmembrane a-helices
in the membrane domain.
cytosolic
domain
2 Ca++
PDB 1EUL
membrane
domain
Muscle SERCA
Active site Asp351, which is transiently phosphorylated
during catalysis, is located in a cytosolic domain, far from
the Ca++ binding sites.
SERCA structure has been determined in the presence &
absence of Ca++, with or without substrate or product
analogs and inhibitors.
 Substantial differences in conformation have been
interpreted as corresponding to different stages of the
reaction cycle.
 Large conformational changes in the cytosolic domain
of SERCA are accompanied by deformation & changes in
position & tilt of transmembrane a-helices.
 The data indicate that when Ca++ dissociates:
• water molecules enter Ca++ binding sites
• charge compensation is provided by protonation of
Ca++-binding residues.
++
Ca
SERCA Conformational Cycle
enzyme
phosphorylation
phosphate
hydrolysis
This simplified cartoon represents the proposed
variation in accessibility & affinity of Ca++-binding sites
during the reaction cycle.
Only 2 transmembrane a-helices are represented, and the
cytosolic domain of SERCA is omitted.
Ca-ATPase
H,K-ATPase
Na,K-ATPase
Sequence comparison of the TM helices in Ptype ATPases of various subfamilies
conserved residues as compared to the
ATP2A1 Ca2+ binding site sequence
non-conserved residues
Topology and architecture of the
catalytic subunits of P-type ATPases.
a strikingly similar fold despite strong sequence
divergence
N-domain binds ATP and serves as a built-in protein kinase, which autophosphorylates the P-domain. The A-domain acts as an intrinsic protein phosphatase
dephosphorylating the P-domain later in the catalytic cycle. The process of
phosphorylation and dephosphorylation is tightly coupled to formation and deformation of
high-affinity transport-binding sites in the M domain by an allosteric mechanism
P-type ATPases involved in neuronal disorders
E1WCa2+ and E2(TG) forms of Ca2+ -ATPase in lipid bilayer
Toyoshima, Nomura, Sugita, FEBS Letters 555 (2003) 106-110
Toyoshima, Nomura, Sugita, FEBS Letters 555 (2003) 106-110
Rearrangements of the transmembrane helices
between E1WCa (violet) and E2(TG) (light green) forms
Toyoshima, Nomura, Sugita, FEBS Letters 555 (2003) 106-110
MacLennan/Green, NATURE, Vol 405, 2000, 633-634
Overview of the structure of the Ca 2+-ATPase
Models of E1 and E2 forms of the a-subunit of
Na,K-ATPase based on the high-resolution structure
of Ca-ATPase (1EUL)
Annu. Rev. Physiol. 2003 .65 : 817–49
Pump Function: Post-Albers Cycle
Apell, J. Rev Physiol Biochem Pharmacol (2003) 150:1–35
Pump Function: Post-Albers Cycle
Ping-Pong Mechanism
backward
Apell, J. Rev Physiol Biochem Pharmacol (2003) 150:1–35
Pump Function: Post-Albers Cycle
Ping-Pong Mechanism
Stoichiometry
Na,K-ATPase: 3 Na+ / 2 K+
H,K-ATPase:
2 H + / 2 K+
SR Ca-ATPase: 2 Ca2+ / 2 H+
Apell, J. Rev Physiol Biochem Pharmacol (2003) 150:1–35
Pump Function: Post-Albers Cycle
Ping-Pong Mechanism
Stoichiometry
Na,K-ATPase: 3 Na+ / 2 K+
H,K-ATPase: 2 H+ / 2 K+
SR Ca-ATPase: 2 Ca2+ / 2 H+
Electrogenicity
Na,K-ATPase:
H,K-ATPase:
SR Ca-ATPase:
+1
0
+2
Apell, J. Rev Physiol Biochem Pharmacol (2003) 150:1–35
The Channel Concept of the Ion Pumps
Apell, J. Rev Physiol Biochem Pharmacol (2003) 150:1–35
Experimental Methods
 Charge movements
by electrophysiological methods
by fluorescence methods
RH421 Method
RH421 Standard Experiment:
reveals electrogenic partial reaction
Apell, Bioelectrochemistry 63 (2004) 149– 156
In the E1 conformation of the Na,K-ATPase the two binding sites which bind
K+ or Na+ ions are always occupied in physiological pH, if not by transported
cations then by H+ ions.
The fluorescence decrease with increasing ion concentrations indicates
import of positive charge into the membrane domain of the ATPases
Apell, Bioelectrochemistry 63 (2004) 149– 156
Hypothetical Model of Cytoplasmic Na+ Binding
Apell, J. Membrane Biol. 180, 1–9 (2001)
Hypothetical
Model
CytoplasmicNa
Na++ Binding
Binding
Hypothetical
Model
ofof
Cytoplasmic
Apell, J. Membrane Biol. 180, 1–9 (2001)
2
Hypothetical Model of Cytoplasmic Na+ Binding
Hypothetical Model of Cytoplasmic Na+ Binding
Apell, J. Membrane Biol. 180, 1–9 (2001)
3
Methods of Investigation
 Charge Movements
by electrophysiological techniques
Experimental Methods
 Charge movements
by electrophysiological methods
by fluorescence methods
 Conformational changes
by fluorescence methods
FITC
Methode
pH and conformation dependence of the 5-IAF
label covalently linked to rabbit a1 Na,KATPase.
Titration in
different
conformations
explains the F
shift during pump
cycle at constant
bulk pH
ATP-dependent Ca2+ uptake, H+ counter
transport, and development of transmembrane
electrical potential at low temperature.
oxonol VI
Pyranine (HPTS)
arsenazo III
[Yu et al. 1994]
Energetics of selected reaction steps from
rabbit kidney Na,K-ATPase