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BMS346 > K channel and B subunit > Flashcards

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
Q

Apical K model

A

Apical- cftr, Enac, CaCl + ?

basolaterol-0 Hsk4, Na/K ATPase, NKCC, K (cAMP acitivated)KvLQT1, KCNE3

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26
Q

Hypothesis from apical K model

A

Apical K channels support Cl secretion

channels on apical all drive CL secretion as Q1 and Hsk4 are basolaterol

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27
Q

Effect of apical ATP NHBE

A

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

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28
Q

Results from adding ATP to control and Paxiline

A

Control- add ATP- stimulates Cl secretion

Paxiline- inhibits Bk- reduces Cl secretion

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29
Q

ATP activated ssc using NI, NT, KD (what are they )

A

Ni- not infected
NT- not target= nonsense sequence
kD- Knocked down

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30
Q

results from NI, NT, KD

A

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

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31
Q

Blocking Bk channels effect on CBF

A

Cultured NHBE- 3.5 days without apical wash
CBF measurements- apical PBS addition
Repeat CBF measurements

32
Q

Why do they leave for 3.5 days?

A

to see impact on ASL- cbf dependent on ASL layer height

33
Q

Add membrane to apical side of the membrane?

A

Restores ASL= control experiment to ensure impact restored ASL height

34
Q

Results of ASL CBF

A

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
Q

Final model

A

Apical- Enac, CFTR, Bk, CaCL

Basolateral- NA/K ATPase, Hsk4, NKCC, K (KvLQT1, KCNE3)

36
Q

What are B subunits and what do they do ?

A

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
Q

KCNE family 1-3,4,5

A

1-3= found in epithelial, all have varying weight
103 to 177 aa
1tm domains
excitable long QT syndrome

38
Q

What do KCNE do ?

A

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
Q

Effect of KCNE1 on KCNQ1 investigation

A

Data looking at impact of overexpressing Q1 alone or Q1 and E1 together

40
Q

What was the method for looking at E1 on Q1

A
  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
Q

What are the results of E1 on Q1

A

Voltage dependence is shifted when E1 is over expressed

change in time voltage dependence

42
Q

KCNE1 KO mice and renal function

A

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
Q

Renal epithelial- Apical and basolateral

A
  • Apical= Na/glucose co transporter

- Basolaterol=Na/K atpase, K, Glucose

44
Q

Expression profile of proteins is not the same

A

Q1- at apical surface- same proximal + same distal tubule
E1= only in proximal
* Not completely overlapping

45
Q

Clearance studies in mice- set up

A

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
Q

Method of clearance study

A
  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
Q

Results of the clearance studies

A

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
Q

Fractional excretion

A

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
Q

What happens in the wt clearance studies in fractional excretion

A

Most absorbed

not secreted

50
Q

What are the results from the KCNE1 KO mouse

A

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
Q

Why does glucose not struggle to be reabsorbed

A

playing role in later part of the proximal part
doesn’t absorb as much glucose at that location
increase excretion of water

52
Q

What is chromanol 293B

A

Inhibitor KCNQ1

Add chromanol to WT and it mimics the KO- FeNa and Cl all increased

53
Q

Is E1 regulating Q1

A

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
Q

What does E1 regulate

A

A chromanol sensitive K channel

likely this was Q1 K channel

55
Q

Summary of Q1 and E1

A

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
Q

What does Q1 inhibitor chromanol have an effect on?

A

Only has an effect on wt animals

therefore KCNE1 regulating a chromanol sensitive K channel

57
Q

Q1KO clearance studies compare the Fe % in the KCNQ KO and WT and what does this show

A

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
Q

Patch studies

A

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
Q

What are the results of the patch clamp?

A

E1 regulating K channel that is not Q1

Doesn’t even look voltage dependent- chromanol sensitive K channel that have E1

60
Q

Summary 3

A

KNCE1 regulating another K channel that is not Q1

There is some evidence that Q1 might play a small role

61
Q

KCNE1 +KCNE2=

A

gastric function

- acid secretion parietal cells

62
Q

Stimulants of channels

A

Ach-M3
Histamine
Gastrin- CCKB- stimulate acid secretion

63
Q

What does secretion of HCL do?

A

digests food in stomach before small intestine

64
Q

What does apical cl channel open?

A

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
Q

KCNQ1 KO and gastric function

A 
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
Q

ammonium pulse technique method

A

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
Q

results of ammonium pulse technique

A

No recovery at all- no K to come back into cell to exchange for H channels

68
Q

Why do parietal cells KO secrete less H?

A

Due to problem with apical K secretion

- ko mice Al

69
Q

Investigation to look at stomach PH

A

-KO mice achlorhydric
-even though higher circulating gastrin level
Use histamine

70
Q

Results of stomach acid with +/+, +/-, -/-

A

Hets= response

Homozygous KO= PH more alkaline, struggling to secrete acid and not as much acid in the stomach as normal

71
Q

KCNE2 KO and GASTRIC function

A

Ammoinum pulse technique - NH4

Histamine stimulated

72
Q

Results of KNCE2 KO

A

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
Q

What is apical K channel mediated by?

A

Q1E2 mediated

74
Q

KO Q1

A

Not sufficient K to be exchanged also PH of stomach is higher than It should be

75
Q

E2 KO

A

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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