Lecture 5

Question 1

Na+/K+ ATPase exchange pump (Sodium Potassium Pump)

Answer 1

Maintains Na+ and K+ gradients across the cell membrane
- Pumps 3 Na+ out of the cell for every 2 K+ pumped in

Consumes energy (ATP–>ADP) - Active Transport

Question 2

Structure of the Na+/K+ ATPase exchange pump

Answer 2

Dimeric structure of one alpha (α) subunit and one beta (β) subunit

Question 3

Human Sodium Potassium Pumps

Answer 3

Humans have 4 α genes:
• ATP1A1 (α1): Ubiquitous expression
• ATP1A2 (α2): Muscle and nervous tissue
• ATP1A3 (α3): Neurons
• ATP1A4 (α4): Testis

Humans have 4 β genes:
• ATP1B1 (β1): Ubiquitous expression
• ATP1B2 (β2): Muscle and brain
• ATP1B3 (β3): lung, testis, skeletal muscle, and
liver
• ATP1B4 (β4): Divergent function in placental mammals (i.e. nuclear); still associates with α subunit in fish, avian, and amphibian species

**Don’t need to memorize gene numbers or types, just know that there are several genes with different expression patterns*

Question 4

Sodium Potassium Pump and RMP

Answer 4

• The sodium-potassium exchange pump is “electrogenic”
• Every transport cycle results in the net extrusion of 1 positive charge
• Thus, the pump contributes to the negative RMP of the cell
• This contribution is -6 to -11 mV

Question 5

R.C. Thomas’ experiment on the Electrogenic Nature of the Sodium Potassium Pump

Answer 5

• Impaled a single large snail neuron with 5
electrodes!
• The Li+ and Na+ electrodes inject those ions into the cell, if one injects the other removes cations to compensate
• Injection of Na+, but not Li+, causes the membrane potential to drop
• Blocking the pump with ouabain blocks the Na+ effect • External K+ is also required

Question 6

Oubain

Answer 6

Na+/K+ ATPase blocker

α3 subunit is 1000-fold more sensitive

Question 7

Rapid Onset Dystonia Parkinsonism (ROPD)

Answer 7

Mutations in α3 subunit

can be artificially created by injecting Oubain into the brain

can also be triggered by stress

Question 8

Why Ca2+ is good

Answer 8

• Binds oxygen atoms
• Causes conformational changes in proteins (good for signalling or activating mechanical processes)
• Vesicle exocytosis, muscle contraction, activating other ion channels)
• Due to toxicity, kept at very low levels in cells, making it ideal as a transient signalling molecule

Question 9

Why Ca2+ is bad

Answer 9

• It precipitates phosphates (CaPO4), which can accumulate and become toxic
to cells
• Cannot be chemically altered for neutralization

Question 10

[Ca2+]in vs. [Ca2+]out

Answer 10

[Ca2+]in

Question 11

Ca2+ Pumps

Answer 11

2 types of Ca2+ pumps
• Plasma membrane calcium ATPase (PMCA)
• Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)

Question 12

PMCA Pump

Answer 12

1 Ca2+ ion pumped out of the cell per cycle (hydrolysis

of a single ATP molecule)

Question 13

Human PMCA Pumps

Answer 13

Humans have 4 α genes
• ATP2B1 (PMCA1): Brain/Ubiquitous – lethal if mutated
• ATP2B2 (PMCA2): Brain and muscle – hearing loss and balance
• ATP2B3 (PMCA3): Brain and muscle
• ATP2B3 (PMCA3): Broad distribution – male infertility
* Don’t need to memorize gene numbers or types*

Question 14

SERCA Pump

Answer 14

2 Ca2+ ions pumped into the SR/ER per cycle

hydrolysis of a single ATP molecule

Question 15

Human SERCA Pumps

Answer 15

Humans have 3 α genes
• ATP2A1 (SERCA1): Muscle contraction
• ATP2A2 (SERCA2): Muscle contraction
• ATP2A3 (SERCA3): Non-muscle, but expressed in
cardiomyocytes
Don’t need to memorize subunit genes or types

Question 16

Speed of Ca2+ Pumps

Answer 16

SERCA (2 Ca2+) and PMCA (1 Ca2+) are sluggish at removing Ca2+

Question 17

Speed of Ca2+ Exchangers

Answer 17

NCX and NCKX exchangers remove Ca2+ much more quickly

Question 18

NCX and NCKX exchangers

Answer 18

• Exchangers do not hydrolyze ATP as energy source for moving ions against
their gradients
• Exchangers consume energy from existing ion concentration gradients to move other ions “uphill” against their gradients

Question 19

NCX: • Na+ Ca2+ exchanger (a.k.a. sodium-calcium antiporter)

Answer 19

NCX uses the Na+ gradient
• 1 Ca2+ out for 3 Na+ in
• Most widely distributed sodium-calcium exchanger

Question 20

NCX can operate in reverse

Answer 20

• Both Na+ and Ca2+ want to get into the cell
• Whichever ion experiences the strongest inward full wins
• In order for Na+ to win, it’s charge x driving force must be greater than that for Ca2+
• Otherwise Ca2+ wins and it gets to go in the cell

Question 21

NCKX: Na+-Ca2+-K+ exchanger

Answer 21

Even better at removing cytosolic Ca2+!!

• Uses sodium and potassium gradients to remove Ca2+
• 4 Na+ in and 1K+ out for 1 Ca2+ out (+74 mV for Ca2+ to move inward!)

Reverses at +75mV

Question 22

NCX Structure

Answer 22

9 transmembrane segments

Question 23

NCKX Structure

Answer 23

11 transmembrane segments

N-terminus is cleaved

Question 24

Cl- transport in Immature Neurons

Answer 24

more [Cl-]in than [Cl-]out

Question 25

Cl- transport in Mature Neurons

Answer 25

more [Cl-]out than [Cl-]in

Question 26

How do you end up with more [Cl-]in?

Answer 26

Cotransporters use the Na+ gradient to | move Cl- into the cell

Question 27

NCC (Na+/Cl- cotransporter)

Answer 27

transports 1 Na+and 1 Cl- into the cell *Electrically neutral*

Question 28

NKCC (Na+/K+/Cl- cotransporter)

Answer 28

transport 1 Na+, 1 K+ and 2 Cl- into the cell *Electrically neutral*

Question 29

NCC in Mammals

Answer 29

have 1 NCC gene

Question 30

NKCC in Mammals

Answer 30

have 2 NKCC genes

Question 31

How do you end up with less [Cl-]in?

Answer 31

Cotransporters that use the K+ gradient move Cl- out of the cell

Question 32

KCC: (K+/Cl- cotransporter)

Answer 32

transport 1 K+ and 1 Cl- out of the cell *Electrically Neutral* If you want to inhibit (hyperpolarize) a neuron via Cl- channel activation, you need KCC to produce [Cl-]in

Question 33

KCC in Mammals

Answer 33

4 KCC genes • SLC12A4(KCC1) • SLC12A5(KCC2) • SLC12A6(KCC3) • SLC12A6(KCC4) *Don't need to memorize subunit numbers or types*

Question 34

Cl- co-transporters and pH regulation

Answer 34

* Na+-dependent Cl-/HCO3- exchange system uses the Na+ gradient to move bicarbonate into the cell and protons out * HCO3- is part of a physiological buffering system crucial in the nervous system, where there is little tolerance for fluctuation in pH CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3- * ↑ in cytoplasmic H+ promotes H+ efflux and HCO3- influx * ↑ in HCO3- then shifts the equitation to the left, further neutralizing cytoplasmic pH