K+ Channels and Beta Subunits

Question 1

1. How do K⁺ channels act to drive Na reabsorption and Cl secretion
2. what is the structure of voltage gated channels? Name an example and where it is expressed.
3. Name 2 Ca activated K+​ channels
4. what is the structure of inward rectifying K+​ channels? Name and example and where it is expressed.
5. what is the structure of 2 pore K+​ channels? Name 2 examples and where they are expressed.

Answer 1

1. it hyperpolarises the cell; the negative Vm sets up the driving force for Na reabsorption and Cl secretion
2. 4 subunits come togehter to create a functional channel; each subunit has 6 TM domains and 1 pore
   e. g. KCNQ1 which is expressed in the proximal tubule
3. SK4 and BK
4. 4 subunits make a functional channel; each subunit has 2 TM domains and 1 pore region
   e. g. ROMK expressed in kidney
5. 2 subunits make a functional channel; each subunit has 4 TM domains and 2 pore regions.
   e. g. TWIK1 and TASK2 that are expressed ubiquitously (thought to be housekeeping genes)

Question 2

1. what is Q1 inhibited by?
2. describe the experiment that Cl currents in nasal scrapings and human bronchial epithelial cells are driven by Q1
3. describe the experiment which examined Ba sensitive currents in WT and CF cells.

Answer 2

1. Chromanol 293B
2. Nasal scrapings and human bronchial epithelial cells were subject to PCR,

ussing chambers measured Vte in the presence of amiloride, IBMX and forskolin. increasing [293B] caused Vte to move closer to zero

SCC was also reduced following 293B treatment but it is not however reduced towards zero - suggests other channels also regulate Cl current; this remaining current is Ba sensitive suggesting that other channels must be K+ channels.

3. IBMX and forskolin cause a large increase in 293B sensitive SSC in WT cells

In CF cells, IBMX/Forskolin caused no change in 293B sensitive SSC, however, these cells still shlow a large Ba sensitive current that is not changed by IBMX/forskolin.

Question 3

1. Name an inhibitor of SK4
2. what is CaCC? What is it activated by?
3. what does CaCC activation result in? How is this different in CF cells? What does this imply?

Answer 3

1. Clotrimazole
2. a calcium activated Chloride channel. Activated by UTP activation of purine receptors
3. CaCC activation causes a negative shift in Vm

CF cells show a much larger negative shift in Vm in response to UTP, implying that CaCC is upregulated in CF as a compensatory mechanism

Question 4

1. what were the effects of the following on UTP sensitive currents?
   a) 293B
   b) clotrimazole
2. What does this imply about CaCC regulation?
3. Describe the upper airway model

Answer 4

1a) no effect
1b) had a negative effect on UTP sensitive SSC
2. CaCC is regulated by SK4
3. A - ENaC, CFTR (regulated by Q1 K currents) and CaCC (that is regulated by SK4 mediated K currents)

BL - Na/K ATPase, NKCC1, SK4 (Ca activated K channel) and KCNQ1

Question 5

1. on what membrane are BK channels expressed?
2. What inhibitor inhibits BK channels?
3. what were the effects in ATP activated Cl currents of adding paxilline to:
   a) the basolateral side
   b) the apical side
4. what was effect of shRNA KD of BK channels on Cl currents
5. What ae the effects of paxilline on ciliary beat frequency?

Answer 5

1. apical
2. paxilline
   3a) no effect
   3b) large reduction in current
3. large reduction in current
4. reduction of beat frequency from 10Hz by 50%

Question 6

name 4 ways in which beta subunits can regulate the alpha subunit

Answer 6

1. sit next to the alpha subunit
2. interract with TM domains
3. line the channel pore
4. interract with cytoplasmic tails

Question 7

1. what condition can E1 mutations in excitable cells lead to? Why do these patients also sometimes experience deafness?
2. how was it shown that E1 enhances the function of Q1?

Answer 7

1. LQTS. Experience deafness due to the impacts of E mutations in ear epithelia
2. xenopus oocytes expressed either Q1, E1 or both; 2 electride voltage clamping was used to measure current

Question 8

1. what does staining for a)E1 and b) Q1 reveal about its expression in the proximal tubule
2. describe the in vivo clearance technique
3. what did this technique reveal about E1 WT and KO mice?
4. what was the effect of c293B on a) WT mice and b) E1 KO mice? What does his suggest?
5. What was the phenotype of the Q1 KO in terms of FE, and currents? What does this imply?

6.

Answer 8

1a) E1 is expressd in the apical membrane throughout the cortex
1b) Q1 is also expressed on the A membrane, however its expression is more restricted than E1

2. mice are anaesthetised. BP measured by cannulating the carotid artery indicates the level of anasthesia
   a heated pad and rectal thermometer maintains the temperature of the anasthetised mouse
   the bladder is cannulated to collect urine for analysis
   jugular vein is cannulated for fluid replacement
3. plasma concentration not different between KO and WT mice
   v. high plasma glucose concentrations found for WT and KO (not understood why); lower in KO
   GFR normal
   FE of Na, Cl and glucose increased in KOs

when repeated, more physiological glucose concentratios obtained. However, there was no difference in the plasma [glucose] or FR of glucose between WT and KO
FE of Na, Cl and H₂O increased in KOs

4a) FE of Na, Cl and water was increased - mimicks phenotype of E1 KO
4b) no effect - Q1 had already been inhibited

* E1 regulates Q1

5. FE of Na or H2O was not affected; 293B sensitive currents were not the same as Q1/E1 currents

*** E1 doesnt regulate Q1, but instead another KCNQ family member.**

Question 9

1. Describe the model for acid secretion on the stomach.
2. Why is an apical K+ channel critical for acid secretion?

Answer 9

1. H2O dissociates into H+ and OH-

OH- combines with CO2 to form HCO3-

HCO3 is exchanged for Cl on the basolateral membrane

Cl is secreted across the apical membrane by a Cl channel

H+ is exchanged for K+ by an apical ATPase

2. it provides the K+ for recycling in exchange for H+

Question 10

1. describe is the ammonium pulse technique?
2. describe the results of the ammonium pulse technique in the Q1 KO
3. What was the phenotype of the E2 KO?

Answer 10

carbacol and histamine was used to stimuate H+ secretion

performed in the absence of Na to measure H+/K+ ATPase function

2. pH recovery in KO was prevented; pH recovery was restored in the presence of Na, highlighting that the problem with H+ secretion lies at the apical membrane
3. E2 KOs were less acidic than WT cells and histamine has no affect (normally causes the pH of WT cells to decrease)

plasma gastrin is higher (compensatory mechanism)

no pH recovery upon ammonium pulsation - same as Q1 KO

* E1 enhances the activity of Q1 in the apical membrane.