Trigger 3: Kidney ion channels and Gordon's disease mechanism

Question 1

name three electroneutral cation chloride cotransporteds

Answer 1

NCC, NKCC2 and KCC

Question 2

NCC

Answer 2

sodium chloride co-transporter

Question 3

NKCC

Answer 3

sodium-potassium chloride co-transporter

Question 4

KCC

Answer 4

potassium chloride cotransporter

Question 5

NCC located

Answer 5

apical membrane of DCT cells (end of nephron near collecting duct)

Question 6

DCT

Answer 6

distal convoluted tubule

Question 7

percentage of NaCL reabsorbed within the DCT by NCC

Answer 7

5-10%

Question 8

NKCC2 location

Answer 8

thick ascending loop of Henle (right hand side on diagram)

Question 9

KCC location

Answer 9

proximal tubule (beginning of nephrite_

Question 10

NKCC2 in the loop of hence absorb how much filtered NaCL

Answer 10

15-20%

Question 11

electroneutral cation chloride cotransporters

Answer 11

define final salt conc i urine, affect blood volume and arterial pressure.

Question 12

electroneutral cation chloride cotransporters can be medically inhibited by

Answer 12

thiazide-type diuretics

Question 13

nephrons filters

Answer 13

20% of blood pumped from he heart (180L per day)

Question 14

how much salt is reabsorbed b the nephron

Answer 14

99%

Question 15

What does WNK stand for

Answer 15

‘with no K (lysine) protein kinase)

Question 16

what are WNK

Answer 16

a family of protein kinases which comprise four members (WNK1, WNK2, WNK3, WNk4)

Question 17

mutations in genes encoding…. and …. can be the cause of PHA2 (Gordon’s)

Answer 17

WNK1 and WNK2 and KLHL3/CUL3

Question 18

catalytic domain of WNK1

Answer 18

N terminus residues 221-479

Question 19

catalytic domain of WNK4

Answer 19

N terminus residues 174-432)

Question 20

which other complex can have mutations which cause Gordon’s?

Answer 20

CUL3-KLHL3 complexes

Question 21

which signalling pathway phosphorylates NCC causing it to open and draw in sodium

Answer 21

WNK1-SPAK/OSR1 kinase signalling

Question 22

where is SPAK expression localised to?

Answer 22

the TAL (thick ascending loop) and the DCT

Question 23

which protein needs to be phosphorylated to activate NCC activity

Answer 23

Thr60

Question 24

A protein kinase …

Answer 24

is an enzyme which modifies other proteins by adding phosphate groups to them (phosphorylation)

Question 25

the human kinome

Answer 25

the part of the genome which encodes kinases (500 protein kinase genes)- around 2% of the

Question 26

up to ...% of human proteins may be modified

Answer 26

30%

Question 27

WNK kinases make up a unique branch of the human kinome... most closely related to the

Answer 27

STE KINASES

Question 28

OSR1 and SPAK are part of the

Answer 28

protein kinase family

Question 29

Woks are .... proteins, consisting of how many aa

Answer 29

large- 2126 aa

Question 30

what anchors and orientates ATP in WNKs

Answer 30

lysine at the N terminus (K233)

Question 31

what does WNK1/4 do to SPAK and OSR1

Answer 31

phosphorylates and activate. | Activation is mediated by T-loop phopshorylation

Question 32

relationship between SPAK and OSR1

Answer 32

68% identical in sequence and possess highly similar (90%) catalytic domain

Question 33

WNKs do not

Answer 33

phosphorylate NCC directly

Question 34

what type of protein kinase is SPAK?

Answer 34

Step20p-related protein kinase

Question 35

WNKs do not phosphorylate NCC directly... they interact with

Answer 35

SPAK/OSRR1 which phosphorylates NCCS

Question 36

when is WNK activated

Answer 36

in response to osmotic stress

Question 37

In response to osmotic stress what does WNK1 do

Answer 37

it phosphorylates and activates SPAK/OSR1 kinases. Which goes onto phosphorylate and regulate the activity of ion co-transporters smh as NCC

Question 38

Ubiquitination is an

Answer 38

enzymatic post-translation modification in which ubiquitin protein is attached to a substrate protein--> sends for destruction in the proteasome

Question 39

outline the ubiquitination system

Answer 39

1) ubiquitin is activated by E1 (ubiquitin -activating enzyme) 2) Ub is then transferred to E2 (ubiquitin carrier protein) 3) E3 ligases bind to target substitute and coordinate covalent attach meant of Ub (from E2) 4) target protein may be monoubiquitinated or polyubiquitinated ) sent to the proteosome for proteosomal destruction

Question 40

WNK is a substrate for which complex

Answer 40

CUL3-KLHL3

Question 41

CUL3-KLHL3

Answer 41

ubiquitinates WNK1/4, meaning it is sent for proteosomal degradation instead of phosphorylating SPAK/OSR1- therefore less NCC channels are activated

Question 42

Normal process

Answer 42

Osmotic potential is normal, there CUL3/KLHL3 ubiquitinates WNK4, sending it for degradation in the proteasome. Therefore SPAK/OSR1 is only lightly activated and not many NCC channels are activated - maintaining balance

Question 43

PHAII process

Answer 43

Mutation in WNK4 or CUL3/KLHL3 prevents degradation of WNK4, therefore there are many WNK4 to phosphorylate SPAK/OSR1- therefore powerful activation of NCC

Question 44

what are the mechanisms that contribute to the development of Gordon's syndrome (1-4)

Answer 44

- genetic modification (e.g. WNK1/4 and CUL3/KLHL3) - protein phosphorylation (activation) - protein ubiquitylation (protesomal degradation) - ion homeostasis

Question 45

activated WNk kinases bind to SPAK to phosphorylate them where

Answer 45

Thr243

Question 46

activates SPAK will then bind to NCC and phosphorylate at

Answer 46

Thr60

Question 47

pNCC

Answer 47

has increased intrinsic activity and increases Na influx

Question 48

where is the catalytic lysin in WNK family members locacted

Answer 48

the glycine-rich B strand 2, rather than the normal position in B strand 3 of the kinase domain

Question 49

How are WINK PK different to other PKs

Answer 49

catalytic lysine is found in glycine-rich b strand 2,rather than 3 of the kinase domain

Question 50

chloride sensing by WNK1 involves

Answer 50

inhibition of autophoshorylation

Question 51

NKCC1 causes what to end the cell

Answer 51

Na, K and 2Cl-

Question 52

NCC causes what to end the cell

Answer 52

Na, Cl

Question 53

NKCC2 causes what to end the cell

Answer 53

Na K 2 Cl-

Question 54

WNK4 has how many exons

Answer 54

19

Question 55

all pathogenic variants for WNK4 are

Answer 55

missense

Question 56

all pathogenic variants in WNK4 have been identified in exons

Answer 56

7 and 17

Question 57

relationship between metabolic acidosis and potassium

Answer 57

Renal acid secretion is influenced by serum K+ and may result from the transcellular shift of K+ when intracellular K+ is exchanged for extracellular H+ or vice versa. In hypokalemia, an intracellular acidosis can develop; in hyperkalemia, an intracellular alkalosis can develop.

Question 58

ROMK channels

Answer 58

an ATP-dependent potassium channel

Question 59

Mutations in WNK4 are missense mutations and cause

Answer 59

loss of function, so that WNK4 loses its ability to suppress NCCT and ROMK.

Question 60

ROMk is predominantly expressed in

Answer 60

thick ascending limb and cortical collecting duct.

Question 61

how CUL3/KLHL3 regulates WNK1/4 pathway

Answer 61

1) E1 becomes activated by ATP, with the addition of a Ub 2) E1 transfers Ub to E2 3) E2 is recruited to E3 which is complexed with the substrate (e.g. WNK) 4) Ub transferred to substrate 5) process repeats until WNK is ubiquitinated enough to be sent for proteasomal degradation

Question 62

CUL3 has ......

Answer 62

E3 ubiquitin ligases