Lecture 9 - K+ channels and epithelial function Flashcards
What do K+ channels do ??
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
Role of K+ channels in the airway
Drive chloride secretion
K+ channel Families
Voltage gated - Kv
Inwardly Rectifying- Kir
Two pore
What is the structure of voltage gated K+ channels ?
4 subunits= 1 channel
4 TM
What is the structure of Inwardly rectifying K+ channels ??
4 subunits = one channel
What is the structure of two pore ?
2 subunits= 1 channel
-2 TM domains with pore region 4 required for a functional channel
Example of Kv channel
KCNQ1/KCNE1
Example of Kir
Kir1.1 (ROMK in kidney)
Example of 2P
TWIK-1, TASK-2
If chloride channels open activation of K+ channels will do what?
increase chloride secretion because you increased the driving force
Role of the basolateral K+channel
Set the membrane potential which provides the driving force
Kv channels - Testing the hypothesis K+ maintains cl- secretion
- KCNQ1 - gene
- KVLQT1 - channel protein
- inhibited by chromanol 293B
- fresh nasal epithelium and human cell line -
- effects not conclusive
Chromanol 293B
blocks a small number not all K channels
- very specific
RT- PCR of RNA - NAsal epithelium
-**KvLQT1 mRNA expressed in upper resp tract epithelial cells ***
- Nasal polyps 3 condtions
CF
NON CF
HBE
Ussing Chamber -
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
Experiment CF vs Non CF
-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
Upper airway - other K+ channels and Cl- channels
- hSK4 - Ca2+ activated K+ channel blocked by clotrimazole
- CaCC- Ca2+ activated Cl- channel activated by UTP
2nd Hypothesis
Ca2+ activated K+ channels support Cl- secretion
Effect of apical UTP on normal and CF Nasal tissue
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
Basolateral K+ channel blockers and UTP - UTP induced SCC experiment
- 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
K+ channel blockers - UTP
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
K+ channel blockers - UTP
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
3rd Hypothesis
apical K+ channels support cl- secretion
Apical K+ channel
BK Channel
SK4
Intermediate conductance ca activated K+ channel
BK channels
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
BK channels
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
BK channels and Cilia beat frequency
- 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
Cilia beat frequency protocol
-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»_space;test to check you’ve disrupted ASL and thats whats disrupted the channel.
BK channels & Cilia Beat frequency
**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
Final Model -
Apical - Enac CFTR CaCC BK (Ca2+ activated)
Basolateral - hSK4 (Ca2+ activated) Na/K atpase NKCC2 KvLQT1/KCNE3 (cAMP activated)
