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K channel and B subunit Flashcards

1
Q

What do K channels do

A

Open k channels
drive Vm to Ek therefore K channels maintain neg vm
just like excitable cells

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2
Q

What is the Ek?

A

~-90mV

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3
Q

K channel families

A
  1. Voltage gated Kv
    - 4 subunits= 1 channel
    - Pore region
    - 6TM spanning domain
  2. Inwardly retifying Kir
    - 4 subunits= 1 channel
    - 1 pore
    - 2 TM, domain
  3. Two pore
    - 2 subunits= 1 channel
    - 2 pore per subunit
    - 4TM spanning
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4
Q

K channels in the epithelia

A

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

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5
Q

Upper airway model hypothesis

A

K maintains Cl secretion

basolateral K channel Important in setting neg membrane pot, DF for Cl secretion

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6
Q

The more negative the membrane potential

A

Bigger DF

increase K and increase Cl and K secretion

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7
Q

Kv channels KCNQ1 (channel protein) inhibition

A

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

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8
Q

RT-PCR of RNA

A

Expression of mRNA
product should be 788 bp
Control as missing step- shouldn’t get any product

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9
Q

Results of PCR RNA

A

Q1K channel expressed in both wt and CF

KvQLT1 mRNA expressed in upper respiratory tract epithelial cells

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10
Q

Ussing chamber work on nasal epithelium

A

VTE plotted against time (2 min periods)
Increase in Vte when you add IBMX

Level out with Fors

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11
Q

Is cAMP activated K channel?

A

Dotted lines are increasing con of IMBX added to basolaterol cell line
shifts closer to O inhibiting transport
blocking K channels- decrease Cl secretion

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12
Q

K channel recycling across the cell line is the amount of

A

through channel controlling Cl channel - indirect change in movement

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13
Q

If you increase 293B or add barium

A

It increase SSC

ba- blocks K channel and brings it down to O

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14
Q

CF verses non-CF- 293B

A 
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

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15
Q

CF verses Non-CF in Ba sensitive ISC bar chart

A

Increase IBMX increase cAMP

Cl secretion driven by barium sensitive K channel on top of Q1 in CF- through additional CL channel

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16
Q

What do we know so far?

A
  • KCNE3- regulates Q1= cAMP activated

- Barium= additional K channel and Cl channels

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17
Q

Upper airway other K and Cl channels

A

HSK4- Ca activated K family - blocked by clotrimazole

CaCC- Ca activated cl channel- activated by UTP

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18
Q

What is the hypothesis for K channels

A

Ca activated K channels support Cl secretion

UTP activates Cl and K- enhances DF for Cl secretion and increase Cl channel activity

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19
Q

Where are the K channels in the upper airway model

A
  • Apical= CFTR, ENAC,CaCl

- Basolateral= Hsk4, KvLQT1, KCNE3 (Na/k ATPase + NKCC)

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20
Q

What is the effect of apical UTP on normal and CF nasal tissue

A

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
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21
Q

What is the effect on basolaterol K channel blockers

A

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

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22
Q

How does Clotrimazole work?

A

Ca activated Cl secretion is inhibited by clotrimazole

indirect via K channel;

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23
Q

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

A

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

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24
Q

Upper airway model 3

A

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

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25
Apical K model
Apical- cftr, Enac, CaCl + ? | basolaterol-0 Hsk4, Na/K ATPase, NKCC, K (cAMP acitivated)KvLQT1, KCNE3
26
Hypothesis from apical K model
Apical K channels support Cl secretion | channels on apical all drive CL secretion as Q1 and Hsk4 are basolaterol
27
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
28
Results from adding ATP to control and Paxiline
Control- add ATP- stimulates Cl secretion | Paxiline- inhibits Bk- reduces Cl secretion
29
ATP activated ssc using NI, NT, KD (what are they )
Ni- not infected NT- not target= nonsense sequence kD- Knocked down
30
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
31
Blocking Bk channels effect on CBF
Cultured NHBE- 3.5 days without apical wash CBF measurements- apical PBS addition Repeat CBF measurements
32
Why do they leave for 3.5 days?
to see impact on ASL- cbf dependent on ASL layer height
33
Add membrane to apical side of the membrane?
Restores ASL= control experiment to ensure impact restored ASL height
34
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
35
Final model
Apical- Enac, CFTR, Bk, CaCL | Basolateral- NA/K ATPase, Hsk4, NKCC, K (KvLQT1, KCNE3)
36
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
37
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
38
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
39
Effect of KCNE1 on KCNQ1 investigation
Data looking at impact of overexpressing Q1 alone or Q1 and E1 together
40
What was the method for looking at E1 on Q1
1. Clamped potentials- 2 electrode voltage clamo of xenopus oocytes expressing xRNA encoding KCNQ1 or KVNQ1 +KCNE1 2. Voltage dependence- gaps between lines increases as you go down currents
41
What are the results of E1 on Q1
Voltage dependence is shifted when E1 is over expressed | change in time voltage dependence
42
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
43
Renal epithelial- Apical and basolateral
- Apical= Na/glucose co transporter | - Basolaterol=Na/K atpase, K, Glucose
44
Expression profile of proteins is not the same
Q1- at apical surface- same proximal + same distal tubule E1= only in proximal * Not completely overlapping
45
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
46
Method of clearance study
1. anesthetise mouse 2. collect urine- different conc of ions and solutes and volume produced over time 3. Blood sample- conc of ions and solutes 4. BP- how viable animal is, low perfusion of the kidney is low (Bad sample)- good for measuring how out the animals are 5. heated pad- monitoring body tempp
47
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
48
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
49
What happens in the wt clearance studies in fractional excretion
Most absorbed | not secreted
50
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
51
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
52
What is chromanol 293B
Inhibitor KCNQ1 | Add chromanol to WT and it mimics the KO- FeNa and Cl all increased
53
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
54
What does E1 regulate
A chromanol sensitive K channel | likely this was Q1 K channel
55
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
56
What does Q1 inhibitor chromanol have an effect on?
Only has an effect on wt animals | therefore KCNE1 regulating a chromanol sensitive K channel
57
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
58
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
59
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
60
Summary 3
KNCE1 regulating another K channel that is not Q1 | There is some evidence that Q1 might play a small role
61
KCNE1 +KCNE2=
gastric function | - acid secretion parietal cells
62
Stimulants of channels
Ach-M3 Histamine Gastrin- CCKB- stimulate acid secretion
63
What does secretion of HCL do?
digests food in stomach before small intestine
64
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
65
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 ```
66
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
67
results of ammonium pulse technique
No recovery at all- no K to come back into cell to exchange for H channels
68
Why do parietal cells KO secrete less H?
Due to problem with apical K secretion | - ko mice Al
69
Investigation to look at stomach PH
-KO mice achlorhydric -even though higher circulating gastrin level Use histamine
70
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
71
KCNE2 KO and GASTRIC function
Ammoinum pulse technique - NH4 | Histamine stimulated
72
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
73
What is apical K channel mediated by?
Q1E2 mediated
74
KO Q1
Not sufficient K to be exchanged also PH of stomach is higher than It should be
75
E2 KO
reduction in K secretion, less exchange of K for H ions so PH is higher than it should be

BMS346 (5 decks)

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