membrane transport na k atpase Flashcards
what type of atpase transports do we have
- P-type ATPases
V-type ATPases - in intracellular organs
proton-pumps
- F-type ATPases
ABC transporters
what char. can you say about P-type atpase? (General characteristics since they also are subdivided to groups)
Integral membrane proteins
Similarities in the structure: T domain – transport N domain – ATP/ADP binding P domain – phosphorylation A domain – phosphatase activity
ATP hydrolysis
- phosphorylation/dephosphorylation of the transporter
what else can you say about P type atpase
• The P type ATPases are a large group of evolutionarily related ion and lipid pumps that are found in bacteria, archaea and eukaryotes
• They are α helical bundle primary transporters referred to as P type ATPases because they catalyse auto- (or self) phosphorylation of a key conserved aspartate residue within the pump
• In addition, they all appear to interconvert between at least two different conformations, denoted by E1 and E2
• Most members of this transporter family are specific for the pumping of a large array of cations, however one subfamily is involved in
flipping phospholipids to maintain the asymmetric nature of the biomembrane (Flippases)
prominent examples of P type atpase
• Prominent examples of P4type ATPases are-
1) The sodium potassium pump (Na+,K+ATPase)
Having alpha 1, 2, 3 or 4 isoforms (located mainly in brain, 3 is in testies I think)
2) The plasma membrane proton pump (H+ATPase).
3) The proton potassium pump (H+,K+ATPase) located in the stomach parietal cells for K+ absorption and H+ excretion
4) The sarco/endoplasmic Ca2+ATPase
Having Isoforms:
A) SERCA1(striatal muscle)
B) SERCA1(smooth muscle and cardiac muscle)
C) SERCA3 (Platelets and endothelial cells).
5) Plasma membrane Calcium pumps atpase, which include Plasma membrane ATPases(PCMA) isoforms:
o PCMA1 General
o PCMA2 Neuronal(higher affinity for cAMP) phosphorylation than PCMA4 cAMP phosphorylation than PCMA4
o PCMA3 striatal muscle, brain
o PCMA4 General
and PMCA 5
6) ATP dependent aminophospholipid translocase
phosphatidyl serine, phosphatidyl etanolamine
asymetric membrane distribution
(the aminophospholipid translocases transport phosphatidylserine and phosphatidylethanolamine from one side of a bilayer to another)
Postulated mechanism of Na K atpase?
- The ATPase pump starts off in the E1-ATP conformation, then 3 Na+ molecules enter the pump from the intracellular environment. This will give the E1-ATP-3Na+ analog
- After the loss of ADP we get the E1-P-3Na+ analog (phosphorylated)
- Then, after the loss of one Na+ to the extracellular space, we get the E2-P-2Na+ analog. The pump has changed shape and is now in the E2 conformation.
The next two Na+ also leave the pump in the extracellular space and get the E2-P conformation. - Next 2 K+ ions will enter the pump, from the extracellular environment, to get the E2-P-2K analog. Then the phosphate leaves as inorganic phosphate Pi and we get the E2-2K analog
- When the phosphate has left, an ATP binds to the pump and we get the E2-2K-ATP analog. After the ATP has added, the pump returns to the E1 conformation E1-2K-ATP analog.
- The 2 K+ ions are pumped into the cytosolic side, after the conformational change, back to E1 and the pump returns to step 1 conformation E1-ATP
• The E1 conformation has a high affinity for Na+ and ATP, and the E2 conformation has a low affinity for ATP, which supports this theory of the mechanism.
Na+ also leave the pump in the extracellular space and get the E21P conformation
4. Next 2 K+ ions will enter the pump, from the extracellular environment, to get the E21P12K analog. Then the phosphate leaves as inorganic phosphate Pi and we get the E212K analog
5. When the phosphate has left, an ATP binds to the pump and we get the E212K1 ATP analog. After the ATP has added, the pump returns to the E1 conformation E112K1ATP analog
6. The 2 K+ ions are pumped into the cytosolic side, after the conformational change, back to E1 and the pump returns to step 1 conformation E11ATP
• The E1 conformation has a high affinity for Na+ and ATP, and the E2 conformation has a low affinity for ATP, which supports this theory of the mechanism
what are the na k atpase domain functions? (T,N,P,A)
T domain – transport
N domain – ATP/ADP binding
P domain – phosphorylation
A domain – phosphatase activity
SERA CA PUMP structure:
The protein is divided into four functional domains despite consisting of only one polypeptide chain. These domains are denoted as M, N, P, and A. Domain M contains the transmembrane section described above as well as the calcium binding sites, and is the only domain of the protein in the lumen of the SR. It consists mostly of α-helices spanning the membrane, barring a few luminal loops. Located in the sarcoplasm, domains N, P, and A associate mostly with ATP hydrolysis. The catalytic site is formed from the association of A and P, the binding site is formed by the N domain, and the phosphorylated residue, Asp-351, is located in the P domain.
M domain- 10 transmembrane segemtns with 2 Ca+ binding sites.
Actuator (A) domain which mediates the movement of the N and P domains during catalytic function.
A nucleotide binding (N) domain with atp binding site.
Phosph
Theory of mechanism of serca Ca+ pump (not sure if needed to know…)
- Ca2+ bind on the transmembrane (M) domain in the two calcium binding sites. ATP binds in the nucleotide (N) binding domain. This will cause a conformational change in the N domain (E1-P conformation)
- The phosphoryl group from ATP is group transferred to Asp351 residue in the phosphorylation (P) domain resulting in ATP -> ADP. This process requires magnesium (E1-P conformation)
- The phosphorylation leads to conformational changes releasing calcium into the lumen (E2-P conformation)
- The activator (A) domain moves causing release of ADP (E2-P conformation)
- The P domain becomes dephosphorylated (E2-P conformation)
- The A domain returns to its original conformation releasing magnesium (E2 conformation)
- P and M domain resets to E1 conformation (E1 conformation)
Na K atpase structure (more detailed…)
The Na+-K+ pump is a P-type ATPase with a structure similar to the H+-K+-ATPase and the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA 3) . Overall, the structure of the sodium-potassium-pump is a transmembrane protein with three subunits labeled α, β, and FXYD.
α-Subunit
The α-subunit is the largest subunit and contains the binding sites for Na+, K+, and ATP. This subunit is composed of 10 transmembrane α-helices (M1-M10). These helices are centered around a three helix bundle formed by M4-M6[1]. The binding sites for K+ and Na+ are located within the transmembrane helices. Additionally, there are three functional domains located on the cytoplasmic face of the membrane: the actuator domain (A), the nucleotide-binding domain (N), and the phosphorylation domain (P)[5]. There are 4 known isoforms of the α-subunit, but even the two most divergent isoforms share 78% sequence identity. The majority of structural diversity among the isoforms occurs at the N-terminus, the first extracellular loop, and the third cytosolic domain. This diversity can influence the rate ion transport and the ability to act as a signaling receptor [4].
β-Subunit
The β-subunit is a single spanning membrane protein with a transmembrane α-helix and a glycosylated extracellular domain [2]. This subunit uses a cluster of aromatic residues to bind to the M7 and M10 helices of the α-subunit within the lipid bilayer. These residues also make contact with a cholesterol molecule, the presence of which is necessary for ion transport to occur. Contact between the α and β subunits also occurs at various residues in the extracellular domains [5]. The β-subunit has important roles in targeting the polypeptide to the membrane and in providing stability. It plays a role in providing binding specificity for potassium ions [5].
FXYD Subunit
The FXYD subunit, sometimes known as the γ-subunit, is an accessory regulatory protein comprised of a transmembrane α-helix and an extracellular domain (which is not shown in this structure)[2]. Regulation of ion pumping action by FXYD has been shown to be tissue and isoform specific [2].
- ATP is hydrolyzed, leading to phosphorylation of the pump at a highly conserved aspartate369 residue and subsequent release of ADP
a bit from lecture about na/ k atpase:
Na,K-ATPase: at least two subunits (αβ), may be three:
- α subunit 4 izoforms
- β subunit 3 izoforms
minimal αβ functional (at least 12 izoenzime compositions – different pharmacological and kinetic chracteristics)
FXYD (N-terminal segment Phe-X-Tyr-Asp motive)
- regulates the transport kinetics of the α-subunit
- not present in all tissues
- its role in the kidney and heart muscle is known
Explain about Oubain (strophantine characteristics)
Ouabain (Strophantine)
Ouabain /wɑːˈbɑːɪn/[1] also known as g-strophanthin, is a plant derived toxic substance that was traditionally used as an arrow poison in eastern Africa for both hunting and warfare. Ouabain is a cardiac glycoside and in lower doses, can be used medically to treat hypotension and some arrhythmias.
- It is a potent inhibitor of the Na+/K+ ATPase pump (Increasing Na+ concentration intracellularily)
- Ouabain involves its binding to and inhibition of the plasma membrane Na+/K+ATPase, especially at the higher concentrations attainable in vitro or with intravenous dosage.
- Inhibition of the sodium potassium pump has a secondary effect on the handling of calcium ions by sodium calcium exchanger (NCX-1Ca out the cell for 3 sodium into the cell, electrogenic). The inhibition of the antiport (which used the electrochemical gradiant of Na++ to tranport ca out of the cell or into the SR) accumalates Ca+ in the cell and produces positive iontropic effect.
- Digoxin is a structurally related and more lipophilic cardiac glycoside that largely replaced Ouabain for therapy because of its superior oral bioavailability.
• Digoxin continues to be used therapeutically for many of the same indications in which Ouabain was used (including atrial fibrillation and congestive heart failure)
localization of the Alpha 1 subunit isofroms of the sodium postassium pump
α1 - all tissues (heart also), kidney outer medulla only α1
α2 - striatal muscle, smooth muscle, heart (T-tubules)
brain (astrocytes)
adipocytes
α3 - brain (neurons)
heart (small amount)
ovary
leukocytes
α4 - testis
- They have a distinct sensitvity to Ouabain inhibition with α2 being the most sensitive ( can be affected at very low concentrations of just 0.1 pM).
- α3 will be sensitive to Ouabain at a higher concentration of 30nM
- α1 will be the least sensitive to Ouabain and is only affected at very high concentrations of 0.1mM.
what are Endogenous cardiac glycosides and what is their char and mechanism.
Endogenous cardiac glycosides:
steroid structure
synthesis in zona fasciculata from progesterone
concentration in the plasma: 10-9 M
role: regulation of the vascular tone (alpha 2 isoform)
they are related In certain forms of hypertension(Salt and volume/dependent – low renin level)
mechanism:
Kidney Na+ loss ↓
↓
[Na+] plasma ↑
↓
Blood volume ↑
? ↓ ?
Release of endogenous cardiac glycosides from the adrenal cortex
↓
Vascular tone ↑
[Na+]i ↑ → Na+- Ca2+ exchange → [Ca2+]i ↑
Sustained treatment with cardiac glycosides → → hypertension
Affinity of ATPASE characteristics:
- The Km (substrate concentration at half maximum velocity( Vmax ) is how efficient the enzyme is for that particular substrate) values of the Na+/K+ ATPase pump are as follows:
1. For K+ (extracellular) the Km is around 0.5mM which is quite low meaning at physiological conditions the K+ binding sites are saturated (High affinity for K+) (serum potassium levels are around 3.5-5~)
2. For ATP the Km is around 0.15mM which is also very low meaning a high affinity for ATP and saturation at physiological conditions usually(except anoxic conditions such as the kidney medulla)
3. The Km for Na+(intracellular) is 10-20mM which is a lot higher than of K+ or ATP. Hence there is a low affinity for Na+
• This means at physiological value of Na+ there will be less than 50%
saturation of the Na+ binding sites. Because of this the pump reacts only to changes in Na+ intracellular concentration!!
On the Citoplasmic C terminal loop-
cAMP-dependent phosphorylation – inhibition of the enzyme
phoshorylation by PKC – increased endocytosis of the enzyme
- Nearly 30% of the total ATP production in the cell is used by Na+/K+ ATPase pumps
random unimportant facts about sodium potassium pump
- Nearly 30% of the total ATP
- In neurons 50% of ATP is used for the pumps
- At normal physiological values of Na+ and K+ the pumps activity is around 15% which means it has a large revese capacity (it can pump a lot more, changing concentractions more acutely untill it reverses its flow)
- In neurons the activity is increased nearly 25 times during an action potential.
K0.5 for ATP is 300 - 800 µM
Anoxia (extreme lack of oxygen)
