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BMS 346- Epithelial physiology in health and disease > Lecture 9 - K+ channels and epithelial function > Flashcards

Lecture 9 - K+ channels and epithelial function Flashcards

1
Q

What do K+ channels do ??

A

High IC K+ conc and low EC

  • Open K + channel
  • Drives Vm to Ek (Nernst)
  • Therefore K+ channels maintain negative Vm
  • ve mem potential important in chloride secretion
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2
Q

Role of K+ channels in the airway

A

Drive chloride secretion

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3
Q

K+ channel Families

A

Voltage gated - Kv
Inwardly Rectifying- Kir
Two pore

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4
Q

What is the structure of voltage gated K+ channels ?

A

4 subunits= 1 channel

4 TM

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5
Q

What is the structure of Inwardly rectifying K+ channels ??

A

4 subunits = one channel

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6
Q

What is the structure of two pore ?

A

2 subunits= 1 channel

-2 TM domains with pore region 4 required for a functional channel

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7
Q

Example of Kv channel

A

KCNQ1/KCNE1

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8
Q

Example of Kir

A

Kir1.1 (ROMK in kidney)

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9
Q

Example of 2P

A

TWIK-1, TASK-2

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10
Q

If chloride channels open activation of K+ channels will do what?

A

increase chloride secretion because you increased the driving force

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11
Q

Role of the basolateral K+channel

A

Set the membrane potential which provides the driving force

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12
Q

Kv channels - Testing the hypothesis K+ maintains cl- secretion

A
  • KCNQ1 - gene
  • KVLQT1 - channel protein
  • inhibited by chromanol 293B
  • fresh nasal epithelium and human cell line -
  • effects not conclusive
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13
Q

Chromanol 293B

A

blocks a small number not all K channels

- very specific

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14
Q

RT- PCR of RNA - NAsal epithelium

A

-**KvLQT1 mRNA expressed in upper resp tract epithelial cells ***
- Nasal polyps 3 condtions
CF
NON CF
HBE

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15
Q

Ussing Chamber -

A

Nasal epithelium -

  • Block KvLQT1 inhibit Cl- secretion
  • All plus amiloride - rid of ENac
  • Adding Chromanol 293 B is inhibiting Transport – blocks K channel > less cl- secretion so transepithelial potential becomes less negative - reducing driving force
  • Cant measure K+ directly using ussing chamber – cl secretion indirect measurement of K+ because you cant meausre K+ movement
  • Cl secretion indriect indicator
  • Increase conc of 293 B inhibit the Scc Blocking K preventing cl secretion

Or Barium – K channel blocker - takes it to zero likely

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16
Q

Experiment CF vs Non CF

A

-KvQLT1 expression normal in CF
-Lack Cl- channels underlies lack of response
-Short circuit currents-
293B sensitive ISC
-Barium sensitive ISC
293B sensitive ISC -
IBMX and Forskolin activating CFTR and CAMP
-CF tissue cAMP channels non functional – no cl- secretion going on

-Barium sensitive ISC –
Increases a little bit in in non CF with IBMX/FOR
In Cf some CL- secretion
-The fact barium had an effect shows there got to be other channels involved

17
Q

Upper airway - other K+ channels and Cl- channels

A
  • hSK4 - Ca2+ activated K+ channel blocked by clotrimazole

- CaCC- Ca2+ activated Cl- channel activated by UTP

18
Q

2nd Hypothesis

A

Ca2+ activated K+ channels support Cl- secretion

19
Q

Effect of apical UTP on normal and CF Nasal tissue

A

Upregulation of cacc in cf - not sufficient to replace all the Cl secretion but alleviates symptoms a little
Hyper polarisation shift in transepithelial potential when UTP added – consistent with stimulating CL- secretion through activated ca channels
Response enhanced in CF cells in SCC

20
Q

Basolateral K+ channel blockers and UTP - UTP induced SCC experiment

A
  • in absence and presence of 23B or Clotrimazole
  • all in presence of Amiloride & NO cAMP stimulation

Ca2+ activated Cl- secretion inhibited by clotrimazole - indirectly via K+ channels
-More cl secretion as we go down the screen in response to Ca
Clotrimazole blocks – HSK4
-  HSK4 and ca channels are linked functionally through changes in IC ca

21
Q

K+ channel blockers - UTP

A

All amiloride and cAMP stimulation

  • adding UTP to cause increase in ISC ca - activation of Q1 and if there CFTR
  • q1 K channel drives cl secretion
  • biggest inhibition when clotrimazole added
  • Ca dependent cl secretion driving force from Q1 and hSK4 channels
  • CF enhanced response to increase in ISC Ca - enhancement of
  • block 293b
22
Q

K+ channel blockers - UTP

A

All amiloride and cAMP stimulation

  • adding UTP to cause increase in ISC ca - activation of Q1 and if there CFTR
  • q1 K channel drives cl secretion
  • biggest inhibition when clotrimazole added
  • Ca dependent cl secretion driving force from Q1 and hSK4 channels
  • CF enhanced response to increase in ISC Ca - enhancement of ca activated cl secretion
  • block 293b now functional because cAMP are up then contributes to driving force through cl channels that are functioning
23
Q

3rd Hypothesis

A

apical K+ channels support cl- secretion

24
Q

Apical K+ channel

A

BK Channel

25
Q

SK4

A

Intermediate conductance ca activated K+ channel

26
Q

BK channels

A

BK channels are large conductance channels – amount of K channels through pore per unit time

  • On apical membrane
  • ATP activates purine receptors and cause increase in ISC ca and activates BK channel
  • Paxilline addition – response to ATP is lost
27
Q

BK channels

A

BK channels are large conductance channels – amount of K channels through pore per unit time

  • Ca activated
  • On apical membrane
  • ATP activates purine receptors and cause increase in ISC ca and activates BK channel
  • Paxilline addition – response to ATP is lost
  • -Paxilline addition bsaolaterally no effect therefore BK apical
  • Used ShRNA and KD BK channel
28
Q

BK channels and Cilia beat frequency

A
  • Leave 3.5 days for impact on ASL
  • CBF dependent on ASL height
  • ## Add medium to apical membrane - Restore ASL - control experiment to ensure impact related to ASL height
29
Q

Cilia beat frequency protocol

A

-Cells grow basolateral down and apical up and insert
-Sit insert in a plate
Cell culture medium in bottom of the well
-Upper airways make there on airway surface layer
PBS- increases ASL – R manually restored height of layer&raquo_space;test to check you’ve disrupted ASL and thats whats disrupted the channel.

30
Q

BK channels & Cilia Beat frequency

A

**Shows paxilline effecting cells ASL and getting it to the right height to function
KO BK KO ability to get ASL right height and cells to secrete cl-
control vs paxilline
cells exposed to paxilline for 3 days CBF low > becuase height of ASL is low because BK blocked so Cl- secretion low affecting ability to set height of liquid layer
- PBS added cause CBF to rise to control level but no effect on control

31
Q

Final Model -

A 
Apical - 
Enac 
CFTR 
CaCC
BK (Ca2+ activated)
Basolateral - 
hSK4 (Ca2+ activated)
Na/K atpase 
NKCC2 
KvLQT1/KCNE3 (cAMP activated)

BMS 346- Epithelial physiology in health and disease (13 decks)

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