β Subunits

Question 1

1. what are β-subunits?
2. give an example of a β subunit found within the kidney and its associated channel

Answer 1

1. additional proteins that interract with and regulate the ion channel
2. Barrtin that regulates CLCK

Question 2

1. what is the resting potential of cardiomyocytes
2. describe the ionic events of the cardiac action potential

Answer 2

1. -80 - -90mV
2. at -60 - -70mV, Na_v channels are activated, leading to sodium influx which drives rapid depolarisation

at -30 - -40mV, Ca_v channels are activated to mediate the plateau phase

Na_v channels undergo fast depolarisation. At the same time I_K and I_Ca increase, in order to reduce the overshoot and to drive the plateau phase respectively

K_v channels are delayed rectifiers therefore become activated slowly. They are fully activated at the end of the plateau phase, where they drive the fast repolarisation

Question 3

Name the 2 K currents that mediate cardiomyocyte repolarisation, and their individual properties

Answer 3

1. I_KR
   - rapid activation and inactivation
   - INWARD rectification
   - delayed rectifier
   - mediated by hERG1
2. I_Ks
   - slow activation
   - outward rectification
   - delayed rectifier
   - mediated by KCNQ1

Question 4

Name 3 K_v beta subunits that are expressed in the heart, and what 2 of them regulate

Answer 4

1. KNCE1 (regulates KCNQ1)
2. KCNE2 (regulates hERG1)
3. KNCE3

Question 5

1. what are the currents produced in CHO cells transfected with E1?
2. what are the currents produced in CHO cells transfected with Q1?
3. what are the currents produced in CHO cells transfected with Q1 and E1?
4. from these findings what can be concluded?
5. In what manner does E1 regulate the Vdep of Q1?

Answer 5

1. no currents
2. small currents
3. significantly bigger currents than with Q1 alone
4. E1 enhances Q1’s activity
5. is causes a positive shift in Vrev by ~30mV, which is similar to the Vdep of I_KS

Question 6

1. what is Long QT syndrome characterised by?
2. what is the effects of the mutations on the cardiac action potential?
3. what does this disrupt?
4. what may this lead to?

Answer 6

1. a depolarisation defect leading to a long QT interval (normal = 0.36 secs, can increase to 0.54secs)
2. an extended plateau phase as K⁺ channels take longer to activate
3. co-ordination of heart contractility - ventricles are unable to depolarise as they are still in the refractory period
4. ventricular tachicardia (>100bpm, with at least 3 irregular beats in a row) and ventricular arryhthmia (which is likely to develop into asystole)

Question 7

Name 4 LQT5 mutations and their effects on I_K

Answer 7

1. V47F - smaller currents and altered Vdep
2. L51H - complete loss of E1 function, as currents are the same as those without E1
3. D76N - no I_K - mutation has negative effect on Q1
4. W87R - smaller currents and altered Vdep

Question 8

Name 3 ways in which E1 regulates Q1

Answer 8

1. Gating (changes Vdep therefore currents are bigger)
2. Trafficking (has a role in the trafficking of Q1 to the membrane)
3. Regulation (tranduces the phosphorylation of Q1 by PKA into bigger currents)

Question 9

TRAFFICKING

1. what was used to tag E1, to enable its expression pattern to be visualised?
2. where was WT E1 found to be localised?
3. where was L51H E1 found to be localised?
4. what was used to show localisation in the ER?
5. Where was Q1 found to be localised when WT E1 was expressed?
6. Where was Q1 found to be localised when L51H was expressed?
7. Why was K_v1.4 used as a control?

Answer 9

1. FLAG peptide, which can be identified by labelled anti-FLAG antibodies
2. cell surface and in the cytosol
3. in thr ER
4. co-staining with calnexin
5. at the cell surface
6. in the cytosol
7. to ensure the effects of the E1 mutation were specifically on Q1, and not Kv channel in general (K_v1.4 is not regulated by E1)

Question 10

1. What is cAMP accumulation mediated activation of I_Ks associated with?
2. how does cAMP accumulation lead to the modification of Q1? By what enzyme is this modification reversed?
3. how is this defective in LQT5 patients, and what does this lead to?

Answer 10

1. the sympathetic nervous system to increase heart rate. Noradrenaline acts on β2AR to activate Gs proteins, which activates adenylyl cyclase leading to cAMP accumulation.
2. cAMP activates PKA, which phosphorylates Q1/E1. This phosphorylation is reversed by protein phosphatase1 (PP1)
3. PKA is unable to phosphorylate Q1/ therefore channel activity isn’t enhanced in response to noradrenaline stimulation, therefore the ventricles can’t keep up with the increased contraction of the atria, leading to ventricular tachicardia.

Question 11

1. What is Okadaic acid and what was it used for in a study into E1 regulation of Q1?
2. How was current measured?
3. What 2 techniques were used to determine phosphorylation by PKA?
4. What phosphomimicking E1 mutation was made?

Answer 11

1. PP1 inhibitor. it is used to maximise E1/Q1 phosphorylation
2. patch clamping
3. ELISA and western blotting
4. S27D

Question 12

1. What were the Q1 currents in cells, and the phosphorylation state of Q1 transfected with E1, Q1 and Yotiao in the absense of cAMP
2. What were the Q1 currents in cells, and the phosphorylation state of Q1 in cells transfected with Q1 and Yotiao in the presence and absense of cAMP?
3. what were the effects of the phosphomimicking mutation on currents?
4. What were the effects of W87R and D76N mutants on Q1 activity on phosphorylation?
5. What were the conclusions of this study?

Answer 12

1. lower currents in the absence of cAMP. Q1 not phosphorylated in the absense of cAMP
2. little difference in currents in the presence/absense of cAMP. Q1 is still phosphorylated in the presence of cAMP
3. enhanced currents in the absense of exogenous cAMP
4. neither interfered with phosphorylation of Q1. both reduced Q1 activity in the presence and absence of cAMP. However W97R E1 didnt inhibit cAMP mediated regulation, while D76N mutant did.
5. E1 is responsible for transducing the Q1 phosphorylation into increased channel activity. the W97R mutant however causes LQT by impacting Q1 in some other way.