lecture 5

Question 1

what are the key steps in epithelial NaCl and water absorption?

Answer 1

1. passive facilitative entry of Na+ across apical membrane down electrochemical gradient
2. active exit of Na+ across basolateral membrane
3. paracellular diffusion of Cl- through tight junctions - forms NaCl through 1 molecule of each
4. osmotically driven water absorption - as osmotic gradient is created

Question 2

what is the pump-leak model?

Answer 2

1. Na+ influx channel - ENaC
2. Na+ pumped out cells across basolateral membrane via Na+K+ ATPase pump
3. causes paracellular transport of Cl- via tight junctions to maintain electroneutrality
4. increases NaCl concentration (lowers water potential) on basolateral side of epithelium, drives osmotic movement of water
5. K+ channels on basolateral membrane maintain membrane potential and recycle K+

Question 3

what is ENaC?

Answer 3

epithelial sodium channel

Question 4

what is the role of ENaC in epithelial tissues?

Answer 4

kidney -> Na+ retention, control of body Na+and water balance, blood pressure control
lung -> Na+ and water absorption, control of amount of ASL and alveolar lining fluid
colon -> Na+ and water reabsorption from diet
sweat gland -> Na+ retention, reabsorption of Na+ by sweat ducts (not followed by water), produces hypotonic sweat secretion

Question 5

what is the structure of ENaC?

Answer 5

-heterotrimer of 3 alpha, beta, gamma subunits coded by 3 genes
-each subunit has 2 transmembrane domains forming pore of channel
-highly selective for Na+
-constantly active but can be regulated
-long extracellular loops are site for regulation by CAPs and SPLUNC1

Question 6

what is an inhibitor of ENaC?

Answer 6

amiloride
-potent, specific inhibitor
-used clinically

Question 7

what is the role of ENaC in the kidneys?

Answer 7

-vital in determining final urinary salt composition through regulatory action of natriueretic hormones (aldosterone) and other paracrines (RAS system)
-aldosterone is important in long term regulation of whole body Na+ and water balance - involves change in ENaC function in aldosterone-sensitive distal nephron

Question 8

what is the aldosterone-sensitive distal nephron (ASDN)?

Answer 8

-last third of the distal convoluted tubule, connecting tubule and the cortical collecting duct

Question 9

what is the function of aldosterone?

Answer 9

-stimulates Na+ reabsorption through ENaC by principle cells in ASDN
-overall effect increased NaCl and H2O absorption which causes blood volume and BP to rise

Question 10

how does aldosterone act on principal cells of the distal nephron?

Answer 10

1. aldosterone enters cells by diffusion and binds to cytosolic receptors
2. once bound, transported to nucleus where aldosterone receptors regulate transcription of certain genes

Question 11

what does aldosterone increase?

Answer 11

1. surface ENaC levels by 2-5 fold
2. Na+/K+ ATPase density
3. ATP supply to support increased Na+/K+ ATPase activity
4. K+ excretion across apical membrane via ROMK

Question 12

how does aldosterone stimulate Na reabsorption?

Answer 12

-increases number of ENaC channels
-by decreasing rate of retrieval/degradation

Question 13

how is ENaC degraded?

Answer 13

1. made in ER
2. goes to trans Golgi
3. stays in membrane for few hours
4. removed to be degraded by proteosomes

Question 14

what is degradation of ENaC regulated by?

Answer 14

ubiquitin ligase (NEDD4-2)

Question 15

how does ubiquitin ligase regulate degradation?

Answer 15

-NEDD4-2 binds t oPY motif and adds ubiquitin group to a lysine residue in N terminal
-ubiquitinisation signals internalisation of ENaC, followed by degradation of ENaC
-alodsterone stimulates production of serum glucocorticoid regulated kinase 1 (SGK1)
-SGK1 phosphorylates NEDD4-2 which prevents binding to ENaC
-inhibits ENaC ubiquitination so ENaC channels increase

Question 16

how is hypertension caused?

Answer 16

-by disregulated ENaC
1. excess aldosterone or mineralocorticoid action
2. liddles syndrome - gain in function of ENaC

Question 17

what is liddles syndrome?

Answer 17

-rare, autosomal dominant genetic disease
-caused by mutations in genes encoding 3 subunits SCNN1a,b,g
-leads to change in cytoplasmic region of ENaC preventing NEDD4-2 binding
-leads to hypokalaemia - low blood K+ levels

Question 18

how is K+ excretion enhanced in Liddles syndrome?

Answer 18

-apical pm is more depolarised, brings in more sodium
-electrical gradient for K+ excretion is increased
-excessive loss of K+ in urine

Question 19

what does blocking ENaC with amiloride do?

Answer 19

-hyper polarises the apical membrane
-reduces K+ loss
-helps prevent hypokalaemia
-lowers BP

Question 20

what is the role of ENaC in the lungs?

Answer 20

-ENaC regulates ASL
-regulates ALF volume for gas transfer
-expressed in apical pm of surface epithelial and alveolar type II cells

Question 21

what regulates ENaC in the conducting airways?

Answer 21

-channel activating proteases eg. CAPS
-anti-proteases
-regulating peptides eg. SPLUNC1
-other local acting factors
-CFTR

Question 22

how do channel activating proteases regulate ENaC?

Answer 22

-by CAPs
-increase ENaC activity via proteolytic cleavage of external loops of alpha and gamma ENaC

Question 23

how do anti-proteases regulate ENaC?

Answer 23

reduce ENaC activity by inhibiting proteases

Question 24

how do regulatory peptides regulate ENaC?

Answer 24

eg. SPLUNC1
reduces ENaC function
secreted into ASL and binds ENaC
protects from internalisation and CAP activation

Question 25

what does inhibiting catches-in B reduce?

Answer 25

fluid absorption across monolayers of primary human airway cells