Download Chem*3560 Lecture 30: Ion pumps in the membrane

Chem*3560
Lecture 30: Ion pumps in the membrane
ATP hydrolysis-driven ion pumps in the plasma membrane and bacterial cell membranes generate the
primary concentration gradients and electrical potentials needed for cell function. Ion-pumping
ATPases fall into three classes (Lehninger p. 417):
P-type ATPase
Na+/K+
Ca2+
Ca2+
H+/K+
H+
Plasma membrane of animal cells
Maintains low Na+ and high K+ inside cells
Maintains membrane potential of –50 to –60 mV
Plasma membrane of animal cells
Maintains cytoplasmic [Ca2+] at about 10–7 M
Endoplasmic reticulum of animal cells
Maintains cytoplasmic [Ca2+] at about 10–7 M
In muscle, stores Ca2+ in sarcoplasmic reticulum so Ca2+ can be
released in response to neuronal signals
Plasma membrane of stomach cells in animals
Acidifies stomach contents
Plasma membrane of plant cells and fungi
H+ gradient used to drive secondary transport
V-type ATPase
H+
Acidifies contents of endosomal and lysosomal compartments in
animal or vacuolar compartments of fungi and higher plants.
Degradative enzymes are activated at the lower pH.
F-type ATPase
H+
Reversible H+ pump in mitochondrial, thylakoid (chloroplast)
and bacterial cell menbranes. If redox reactions of electron
transport produce a high enough electrochemical gradient of H+,
F-type ATPase can condense ADP and Pi to make ATP (ATP
synthase action).
V and F-type ATPases share several aspects of structure. The ATP synthases are more completely
described as Fo F1 -ATPases. F is derived from the term “Coupling Factor of oxidative
phosphorylation”. The F-type ATPase divides into two distinct components (Lehninger p. 418):
Fo is a transmembrane protein consisting of a , b and c subunits arranged as ab2 c 10 . The
isolated Fo forms a passive H+ pore through the membrane which is blocked by the antibiotic
oligomycin. The 'o' subscript stands for 'oligomycin sensitive'.
F1 is a peripheral membrane protein consisting of α 3 β 3 γδε which functions independently as
an ATP hydrolyzing enzyme with three catalytic sites associated with the α 3 β 3 hexamer that
surrounds the central γ subunit.
Each c subunit consists of a
hairpin of two helices, and ten
of these form a cylindrical
bundle about 55 Å in diameter
that traverses the bilayer, with
10 inner helices and 10 outer.
The a subunit covers about
one quarter of the side of the
cylindrical bundle, and
provides the H+ channel.
However the entry channel is discontinuous , and stops halfway across the bilayer. At this point, H+
protonates a critical Asp side chain on the nearest c subunit. There is another half-length channel
that exits from subunit a on the other side of the bilayer, but it is offset from the entry channel. In order
for H+ to complete its passage across the bilayer, the cylinder of c subunits must rotate 40o so that the
AspH+ can line up with the exit half channel and deprotonate. In effect, the Fo module is a
nanoscale turbine powered by H+ moving down its electrochemical gradient, or a pump if the
direction of rotation is reversed (Lehninger pp.680-683).
The F1 module lies on the interior side of the bilayer, and inserts one end of its central γ subunit into the
recess in the middle of the c 10 cylinder of Fo . The ε subunit helps anchor γ to the c 10 cylinder. The b2
pair binds on the outside of the a subunit, and projects well beyond the bilayer where it links up with δ
to hold the α 3 β 3 module in a fixed position. This means α 3 β 3 stays fixed in one orientation, while the
central γε subunits rotate along with c 10 cylinder.
Fluorescence experiments confirm the rotary motion
A fluorescently labelled actin fibre was attached to the exterior facing side of the c 10 cylinder while the
α 3β 3 module F1 was attached to a glass microscope slide. When provided with ATP, rotation could
be observed directly. The angle of rotation per ATP hydrolysed was also determined by these
experiments (Lehninger p.683, Fig. 19-24).
When Fo and F1 associate together in the absence of an electrochemical H+ gradient, the combination
hydrolyses ATP and pumps H+ through Fo towards the exterior face of the membrane. This is actually a
normal role when bacteria grow anaerobically, because the H+ gradient is not being provided by
electron transport, but is still needed for symport of nutrients.
In mitochondria, ATP hydrolysis only occurs if membrane integrity is lost.
In the presence of the H+ gradient produced by electron transport (typically about 150 mV, –ve inside,
and a 3-5 fold higher concentration outside) passage of 3 H+ drives the condensation of ADP and Pi to
yield 1 ATP. This happens in bacteria grown aerobically and is the normal mode of action in
mitochondria. Each H+ causes γ . ε .c 10 to rotate by 40o , making 120o rotation per ATP made.
Not coincidentally, there is a different αβ catalytic pair every 120o in the F1 module.
How does rotary motion provide energy for Pi to condense with ADP?
Since the γ subunit is not symmetrical, it interacts differently with each αβ member of the α 3 β 3
hexamer, so each αβ catalytic unit is in a different conformational state. One unit is in a so called
open state O (very loose binding of substrate), the second is in a loose binding state L, and the third
is in a very tight binding state T, where the binding energy between nucleotide and protein is
very high. The T state site always contains a bound nucleotide.
Step 1: ADP and Pi bind into the loose site L.
Step 2: 3 H+ pass through Fo and this causes the γ.ε.c
γ.ε 10 core to rotate by 120o. The
conformation of the sites change, driven by the energy made available by 3 H+. The original tight
T site becomes O open, so that the ATP contained within it can be released.
Step 3: The original L site becomes T. High binding energy can now drive the condensation
reaction to make ATP. However, because of tight binding, the new ATP can't be released.
All three steps repeat, but are reoriented by 120o for each catalytic cycle
The original O open site has now become an L loose-binding site, and binds the next ADP + Pi .
This is a repeat of step 1, but takes place at the 8 o'clock position instead of the 12 o'clock position due
to the reorientation of the γ subunit (yellow oval shown in the centre of the complex).
The next step 2 loosens the ATP that was made at the previous step 3, allowing it to be
released.
ATP hydrolysis reverses the direction of rotation, and pumps H+ ions out
When the H+ gradient is absent, ATP can enter the open O site, where it is hydrolysed. The hydrolysis
energy is absorbed as binding energy (reverse of step 3).
This changes the conformation and causes a 120o reorientation of the γ subunit (reverse of step 2).
Because γ is connected to c 10 , the c 10 cylinder rotates 120o backwards, so that each c subunit picks
up H+ from the “exit” half channel and releases it in the “entry” half channel, allowing 40o of rotation per
H+. As a result, 3 H+ are pumped from the interior to the
exterior.
Na+/K+ ATPase has two conformations
The overall structure is ααββ. The α subunits (1000 amino
acids) have 8 transmembrane helices each, plus a large
cytoplasmic ATPase domain. Each α subunit appears to be a
complete functional unit, and the purpose of the dimer and the
β subunit (300 amino acids, mostly exposed on the exterior) is
not clear at this time.
1) The starting conformation has three high affinity binding sites
for Na+ accessible from cytoplasm (Lehninger p. 421).
2) When 3 Na+ bind, the α subunit binds ATP, and uses it to
phosphorylate Asp 376, to form aspartyl phosphate.
3) This reaction switches the α subunit’s conformation so that
the Na+ sites have low affinity and are accessible to the
exterior, so that Na+ is released.
4) Now, 2 K+ sites become exposed to the exterior.
Hydrolysis of the aspartyl phosphate occurs, so that the
enzyme returns to its original conformation, with binding sites
accessible to the cytoplasm.
5) The affinity for K+ decreases, so K+ is released, and the
cycle can resume from step 1.