Na/K-ATPase

Question 1

stoichiometry:

net charge carried

Answer 1

3Na (out) :2K (in)

one positive charge OUT of the cell&raquo_space; outward current, ~6mV

Question 2

at any one membrane potential, what does the size of pump current depend on?

Answer 2

the intracellular [Na]

Question 3

at what extracellular (??? probably intraclelular - review) [Na] is the half-maximal pump current? How does this relate to intracellular Na conc?

Answer 3

~10mM

intracellular [Na] = 110-15mM, so the pump can respond to changes sensitively

Question 4

what is extracellular [K] for half-maximal pump current? its significance?

Answer 4

half-max current is at ~1.5mM [K]extracellularly. but serum [K] is ~4mM, so this is not a rate determining factors normally

Question 5

what ATP levels for half-max current?

Answer 5

half-max at ~150microM. physiological [ATP]~mM, so not a rate limiting factor normally

Question 6

describe the pump structure

Answer 6

Three subunits: alpha, beta, gamma.

ALPHA subunit:
contains the catalytic and ion-binding sites, main pump function, receptors for inhibitors (digoxin, oubain)
10 transmembrane alpha-hrlices (M1-M10)
three additional domains on the cytoplasmic face of the membrane:
actuator domain (a),
nucleotide-binding domain (N),
phosphorylation domain (P)

alpha subunit has four isoforms

Question 7

describe the beta subunit

Answer 7

smaller than alpha
regulates the transport of pumps into the plasma membrane and conformational changes
has three isoforms

Question 8

describe gamma subunit

Answer 8

not required for pump function
in is an accessory regulatory protein comprising a transmembrane alpha helix and an extracellular domain (the domain is known as phospholemman, the extent of its phosphorylation determines pump activity)

Question 9

describe the pumping sequence

Answer 9

1. starts in conformation called E1 when three Na ions bind into a pocket in the transmembrane domain of the protein from the cytoplasmic entryway
2. the enzyme catalyses its own phosphorylation by ATP at a conserved aspartate residue, which shuts the ccytoplasmic entryway
3. following hte release of ADP to the cytoplasm, the conformation of the protein then changes to E2, results in the extracellular path opening and a decrease in the affinity for the three Na ions that are bound. Na ions are released into the extracellular side
4. Two extracellular K ions bind the pump while it is still in the E2 conformation
5. this binding leads to the closure of the extracellular entryway and depohsphorylation, which causes the protein to return to the conformation E1
6. upon return to E1, the affinity for Na ions is restored, and for K ions it is reduced. ATP binds and the two K ions are released into the cytoplasm

Question 10

Intracellular pH. what does it suggest|?

Answer 10

Intracellular pH is about 7.2, an order of magnitude greater that if H+ ions were passively distributed. This implies the presence of an active transport.

NHE is activated when the cytosol becomes more acidic.

Question 11

what is the relation of Na and contractile force and what are the underlying mechanisms?

Answer 11

There is a close, steep relation between intracellular [Na] and contractile force.
An increase in intracellular [Na] will cause the reversal potential for NCX to become more negative. Therefore, during the AP, the NCH will spend more time in the reverse mode than under normal intracellular [Na], so this will result in more Ca influx and less Ca efflux.
Subsequently, contraction will increase because of two factors:
1) Increasing Ca influx allows the SR to fill up with more Ca which is available forrelease at the next beat.
2) The resting [Ca]i will increase (with minimal effect on resting force), so that the same increment in Ca released from the SR will produce greater force due to the sigmoidal nature of the relation between [Ca]i and force.

Question 12

how does pH affect contraction force?

Answer 12

The major effect of intracellular pH on contraction is due to the modifications in the binding of Ca to the troponin complex.
	In acidotic conditions, affinity of troponin C for Ca DECREASES, which leads to decreased crossbridge formation producing less force.
	Increasing pH (more alkaline) will shift the force vs [Ca] relationship to the left so that a lower [Ca]i is required to obtain the same force, it also decreasess the threshold [Ca] required for contraction.

Question 13

what is the relationship between pH and [Ca]i?

Answer 13

There is a relationship between pH and [Ca]i.
In the cytosol, as pH becomes more alkaline, [Ca] decreases, and when pH falls, [Ca] increases.
This is due to several reasons:
H+ and Ca share common buffering sites: greater [H+] means more H+ will bind these sites and will release Ca.
Acidosis activates Na/H exchanger and Na/HCO3 symport, leading to an increase in [Na]i, which drives NCX to more Ca influx.
There may also be H+ activated release of Ca from mitochondria.

Question 14

structure of NHE1

Answer 14

N terminal region that has 12 transmembrane areas. its function is related to:

• ion translocation
• inhibitor (eg amiloride) binding
• H+ sensor that promotes activation when pH is decreasing

C-terminal: is cytoplasmic. contains:

• regulatory sites for phosphorylation
• H+ sensor

there are 9 isoforms of NHE