K channel and B subunit Flashcards
What do K channels do
Open k channels
drive Vm to Ek therefore K channels maintain neg vm
just like excitable cells
What is the Ek?
~-90mV
K channel families
- Voltage gated Kv
- 4 subunits= 1 channel
- Pore region
- 6TM spanning domain - Inwardly retifying Kir
- 4 subunits= 1 channel
- 1 pore
- 2 TM, domain - Two pore
- 2 subunits= 1 channel
- 2 pore per subunit
- 4TM spanning
K channels in the epithelia
Kv- KCNQ1/E1-Es, E3, KCNA10- eg Sk4, Bk
Kir (inwardly rectifying)- Kir1.1 (RomK- kidney)
2P- Twink-1, Task-2- K channels and respiratory epithelia
Upper airway model hypothesis
K maintains Cl secretion
basolateral K channel Important in setting neg membrane pot, DF for Cl secretion
The more negative the membrane potential
Bigger DF
increase K and increase Cl and K secretion
Kv channels KCNQ1 (channel protein) inhibition
Inhibited by chromanol 293B (specific inhibition that blocks smaller number of K channels)
pharmacological tool
effects channel 293B are not conclusive but linked with KCNQ1 expression studies
RT-PCR of RNA
Expression of mRNA
product should be 788 bp
Control as missing step- shouldn’t get any product
Results of PCR RNA
Q1K channel expressed in both wt and CF
KvQLT1 mRNA expressed in upper respiratory tract epithelial cells
Ussing chamber work on nasal epithelium
VTE plotted against time (2 min periods)
Increase in Vte when you add IBMX
Level out with Fors
Is cAMP activated K channel?
Dotted lines are increasing con of IMBX added to basolaterol cell line
shifts closer to O inhibiting transport
blocking K channels- decrease Cl secretion
K channel recycling across the cell line is the amount of
through channel controlling Cl channel - indirect change in movement
If you increase 293B or add barium
It increase SSC
ba- blocks K channel and brings it down to O
CF verses non-CF- 293B
293B sensitive ISC control and IBMX (secretion dependent on K channel we're looking at stimulate cAMP)- for non CF and CF Non CF - low 293B sensitive ISC in control - increases with IBMX
CF
- very low in control
- same in IBMX
*Lots of additional cl secretion when you add cAMP in 293B
in CF the cl channels are non functional so when the QIK channels cant drive cAMP dependent secretion
CF verses Non-CF in Ba sensitive ISC bar chart
Increase IBMX increase cAMP
Cl secretion driven by barium sensitive K channel on top of Q1 in CF- through additional CL channel
What do we know so far?
- KCNE3- regulates Q1= cAMP activated
- Barium= additional K channel and Cl channels
Upper airway other K and Cl channels
HSK4- Ca activated K family - blocked by clotrimazole
CaCC- Ca activated cl channel- activated by UTP
What is the hypothesis for K channels
Ca activated K channels support Cl secretion
UTP activates Cl and K- enhances DF for Cl secretion and increase Cl channel activity
Where are the K channels in the upper airway model
- Apical= CFTR, ENAC,CaCl
- Basolateral= Hsk4, KvLQT1, KCNE3 (Na/k ATPase + NKCC)
What is the effect of apical UTP on normal and CF nasal tissue
upregulate CaCC in CF mice- no lung problems as CaCL secretion is great
- add UTP= stimulate PGE receptors stimulating cAMP
- Added in UTP- hyperpolarising shift in VTE= increase secretion
What is the effect on basolaterol K channel blockers
All amiloride and No cAMP stimulation
UTP induced in the absence or presence of 293B or clotrimazole
- 293B= as cAMP is low, Q1 channels don’t function
- Clotrimazole= blocks Hsk4- increase IC ca, response missing, needs Hsk4 to work to drive Cl secretion
How does Clotrimazole work?
Ca activated Cl secretion is inhibited by clotrimazole
indirect via K channel;
K channel blockers- all amiloride and cAMP stimulation (UTP and cAMP)- response to UTP stimulates cAMP and add UTP to increase rise in IC Ca
Ca stimulates increase in SSC
- 293B- Q1 drive secretion, active and cAMP high
-Ca dependent cl secretion has a big role from Hsk4
CF patients
- enhanced Cl secretion
- 293B contributes to DR for move in CF
- DF cl secretion comes from Ca activated Cl channels
Upper airway model 3
Apical- CFTR,ENAC,CaCl
Basolaterol- K (KvLQT1, KCNE3), Na/K ATPase, NKCC
- Q1 CFTR pairing through cAMP
*high cAMP can drive Cl secretion through Ca activated Cl secretion pathways
- Hsk4 and Ca activated channels through Ca
Apical K model
Apical- cftr, Enac, CaCl + ?
basolaterol-0 Hsk4, Na/K ATPase, NKCC, K (cAMP acitivated)KvLQT1, KCNE3
Hypothesis from apical K model
Apical K channels support Cl secretion
channels on apical all drive CL secretion as Q1 and Hsk4 are basolaterol
Effect of apical ATP NHBE
ATP activated ISC - for control and Paxilline (Bk channel blocker, no effect basolaterally)
Bk channel on apical membrane has large conductance Ca activated
ATP activates BK channel
Results from adding ATP to control and Paxiline
Control- add ATP- stimulates Cl secretion
Paxiline- inhibits Bk- reduces Cl secretion
ATP activated ssc using NI, NT, KD (what are they )
Ni- not infected
NT- not target= nonsense sequence
kD- Knocked down
results from NI, NT, KD
NT- no effect in comparison to control
KD- Bk channel and look at the impact on CL channel, reduces secretion through Ca activated Cl pathway channel
Blocking Bk channels effect on CBF
Cultured NHBE- 3.5 days without apical wash
CBF measurements- apical PBS addition
Repeat CBF measurements
Why do they leave for 3.5 days?
to see impact on ASL- cbf dependent on ASL layer height
Add membrane to apical side of the membrane?
Restores ASL= control experiment to ensure impact restored ASL height
Results of ASL CBF
T= let cells set their own height and looked at beating
- control and paxiline
Paxilline= lowers CBF as height of ASL halved, Add PBS and the paxiline CBF rises to control
In Bk KD cells- non existent @T= effective KD
- restore CBF KD- manually add liquid
Final model
Apical- Enac, CFTR, Bk, CaCL
Basolateral- NA/K ATPase, Hsk4, NKCC, K (KvLQT1, KCNE3)
What are B subunits and what do they do ?
Cytoplasmic/ integral membrane proteins
Example- KCNE family- Bartin
Modify properties of a subunit
voltage gated K channel interconnected with helices in a subunit- B change properties with their interaction
KCNE family 1-3,4,5
1-3= found in epithelial, all have varying weight
103 to 177 aa
1tm domains
excitable long QT syndrome
What do KCNE do ?
All regulate Q1K channel and the interaction Es properties of K channel- different
different subunits have different effects on channel- some combos remove voltage dependence of K channel or determine how K channels function under normal condition
Effect of KCNE1 on KCNQ1 investigation
Data looking at impact of overexpressing Q1 alone or Q1 and E1 together
What was the method for looking at E1 on Q1
- Clamped potentials- 2 electrode voltage clamo of xenopus oocytes expressing xRNA encoding KCNQ1 or KVNQ1 +KCNE1
- Voltage dependence- gaps between lines increases as you go down currents
What are the results of E1 on Q1
Voltage dependence is shifted when E1 is over expressed
change in time voltage dependence
KCNE1 KO mice and renal function
real evidence E1 expressed in the kidney proximal tubule
model of glucose handling
Immunostaining- ring of apical membrane containing stained KCNE1 and nucleus
ring of Protein E1 found in the apical membrane, regulating apical K channel likely to be Q1 K channel
Renal epithelial- Apical and basolateral
- Apical= Na/glucose co transporter
- Basolaterol=Na/K atpase, K, Glucose
Expression profile of proteins is not the same
Q1- at apical surface- same proximal + same distal tubule
E1= only in proximal
* Not completely overlapping
Clearance studies in mice- set up
Mouse on back on heated pad- measure temperature/ rectal temperature
- cannulate bladder= collect urine for analysis
- cannulate jugular vein= fluid replacement
- cannulate carotid artery= BP measurement and blood sample
Method of clearance study
- anesthetise mouse
- collect urine- different conc of ions and solutes and volume produced over time
- Blood sample- conc of ions and solutes
- BP- how viable animal is, low perfusion of the kidney is low (Bad sample)- good for measuring how out the animals are
- heated pad- monitoring body tempp
Results of the clearance studies
Plasma levels not sufficiently different
- GFR- smaller than clearance studies in humans, not significantly different, E1 KO has no effect
- Plasma glucose- E1 KO significantly decreased- scale different, lower buts its above normal
Fractional excretion
How much is excreted per unit of time/ how much is filtered over time
100%- everything filtered is secreted
50%- half of whats filtered is secreted
What happens in the wt clearance studies in fractional excretion
Most absorbed
not secreted
What are the results from the KCNE1 KO mouse
Struggle to reabsorb Na, Cl, glucose and fluid
indicate changes in tubular function
losing E1 impacted at the nephron
defect @ proximal tubule- cant reabsorb normally
*glucose is the exception- not struggling to reabsorb
Why does glucose not struggle to be reabsorbed
playing role in later part of the proximal part
doesn’t absorb as much glucose at that location
increase excretion of water
What is chromanol 293B
Inhibitor KCNQ1
Add chromanol to WT and it mimics the KO- FeNa and Cl all increased
Is E1 regulating Q1
Chromanol 293B inhibitor
animals infused in fluid chromanol 293B- block Q1 but not cause them any major problem
- Fe Na= shift fractional excretion of the Na Looks like an E1 KO- if E1 not working when you block Q1 there should be no change in response
*same for Cl and water
What does E1 regulate
A chromanol sensitive K channel
likely this was Q1 K channel
Summary of Q1 and E1
KCNE1 important in Na, Cl, HCO3 and therefore water handling
given location data probably PT, might also have a role more distally
lack of effect on glucose suggests PT (little glucose uptake)
Maintenance membrane potential and therefore transport function
What does Q1 inhibitor chromanol have an effect on?
Only has an effect on wt animals
therefore KCNE1 regulating a chromanol sensitive K channel
Q1KO clearance studies compare the Fe % in the KCNQ KO and WT and what does this show
Some differences under glucose loading
BUT
this Is different to KCNE1 KO mice
* if regulated by E1 you would expect the Fe to be up but there is no difference- suggest Q1 and E1 are separate
Patch studies
Total whole cell current
- decrease in current= chromanol sensitive current
Extracted Q1/E1 K currents should look like from current time graph- increase voltage= it increases
What are the results of the patch clamp?
E1 regulating K channel that is not Q1
Doesn’t even look voltage dependent- chromanol sensitive K channel that have E1
Summary 3
KNCE1 regulating another K channel that is not Q1
There is some evidence that Q1 might play a small role
KCNE1 +KCNE2=
gastric function
- acid secretion parietal cells
Stimulants of channels
Ach-M3
Histamine
Gastrin- CCKB- stimulate acid secretion
What does secretion of HCL do?
digests food in stomach before small intestine
What does apical cl channel open?
allows Cl secretion
Cl bicarbonate secretor
secretion of Cl by cl and K atpase
*not enough K in stomach to support this- has to secrete K across apical membrane to support this
KCNQ1 KO and gastric function
Ammonium pulse technique- NH4 acid load cells look at rate of H excretion stimulate carbachol or histamine Absence Na measurements H/K ATPASE function
ammonium pulse technique method
Ammonia into cells combine with hydrogen ions- alkalisation increase in Ph Then take away ammonia from outside- release H ions and an acidification
- look at how quickly the cell recovers- how quickly the H are secreted
- Absense of Na allows only looking H/K ATPase
results of ammonium pulse technique
No recovery at all- no K to come back into cell to exchange for H channels
Why do parietal cells KO secrete less H?
Due to problem with apical K secretion
- ko mice Al
Investigation to look at stomach PH
-KO mice achlorhydric
-even though higher circulating gastrin level
Use histamine
Results of stomach acid with +/+, +/-, -/-
Hets= response
Homozygous KO= PH more alkaline, struggling to secrete acid and not as much acid in the stomach as normal
KCNE2 KO and GASTRIC function
Ammoinum pulse technique - NH4
Histamine stimulated
Results of KNCE2 KO
All 3 lines superimposed on top of each other in the absence of Na
WT- Recover, secrete H ions
Hets- secrete some acid
No E2- no recovery from acid load- protein K ATPase cant work so no K secretion as E2 isn’t there to regulate Q1
What is apical K channel mediated by?
Q1E2 mediated
KO Q1
Not sufficient K to be exchanged also PH of stomach is higher than It should be
E2 KO
reduction in K secretion, less exchange of K for H ions so PH is higher than it should be
