Dr Neil Marrion

Question 1

Delayed rectifier

Answer 1

Delay before activating
Rectifies away from linear

No steady state inactivation (no beta1 subunit)

Responsible for repolarising the AP
Inactivation = lengthens the AP

Cole-Moore effect = shortens delay with depolarising burst

Question 2

A current

Answer 2

Transient (~10ms) + inactivating due to beta1 subunit

Sets frequency of AP

Activated by AHPs - opposes the progressive depolarisation

Blocked by 4-AP

Question 3

Where does TEA bind?

Answer 3

Tyrosine in the P loop

Question 4

M current

Answer 4

Terminates short bursts of AP firing

Never inactivates - see pure M current by holding Vm @ 30mV (all open/inactivate except M)

Activated by somatostatin receptors - endogenous anti-epileptic

• SST4 K/O = increased seizure sensitivity
• SST4 = inhibits epileptiform activity in the CA1 region via enhancing the M current
• SST4 = novel target for anti-epileptic drugs

Inhibited by mAChRs = slow EPSP
Phasic firing –> burst firing due to inhibiting the M current

Enhance - retigabine + ICA73 = anti-epileptic
Inhibit = Linopiridine - cognitive enhancer - treat vascular dementia + age-related cognitive decline

Question 5

Retigabine

Answer 5

Enhances M-current - physically stops the channel from closing (allosteric hinderance) = anti-convulsant

Moves the channel to more hyperpolarised currents - more sensitive to current changes = cell is less excitable - stops APs firing due to enhanced M current

Possibility of a CNS selective effect - not in heart!

Binds to a tryptophan residue on the bottom of S5

Require 1/4 subunits to bind retigabine to be sensitive to produce full enhancement (concatenation studies)

Failed clinical trials = turned patients blue
ALS = decrease motor excitability
(ALS - familial [SOD + ALS2] + sporadic [EAAT1/2 down-reg, decreased Ca buffers, ADAR2 reduction)

Question 6

ICA73

Answer 6

Enhances M-current = anti-convulsant

Binds on S3 + targets residues in S4 = involved in gating charge (movement of arginine residues in S4) - fixes the channel in the open state

Need 4x ICA73 to bind to be fully sensitive
(concatenation studies)

Much stronger than retigabine

Question 7

Linopiridine

Answer 7

Inhibit M current = cognitive enhancer

Treat vascular dementia + age-related cognitive decline

Question 8

BK Channels

Answer 8

Slo channels 
Calcium-activated K+ channel
Big conductance = 250pS
Voltage + Ca dependent = Po increases with depolarisation + increasing [Ca]
Less Ca sensitive than SK  

fAHP <5ms
Quickens the speed of repolarisation

Weird structure:
7 TMD (EC N-terminus)
4 IC hydrophobic domains
RCK1 domain
RCK2 domain - contains Ca bowl

Inhibitor = Paxilline

Physically tethered to N-type Ca channel

Strong, positive cooperativity = need 4 Ca bowls for fully functional BK - but can opening with 1 bowl (and 0 at very high micomolar concentration due to RCK2)

Important for:
Daily expression of BK channels - increased at nighttime to suppress high frequency firing rate

Question 9

BK + AB42

Answer 9

AD-model mice
IC injection of AB42 suppresses BK channel function = therefore slows down repolarisation, broadens AP spike, less excitable
Inject channel opener = ameliorate deficits via restoring BK channel activity

Question 10

Cole-Moore effect

Answer 10

Delayed rectifier - strong hyperpolarisation preceding a depolarising shift delayed the rise of the K+ current = delay reflects the time required to refill the membrane with K+ after the ions are swept out of the axoplasm via hyperpolarisation

Question 11

4-AP

Answer 11

Blocks A current

Question 12

Which current never inactivates?

Answer 12

M current

Question 13

BK Channel Blocker

Answer 13

Paxilline

Question 14

A current blocker

Answer 14

4-AP

Question 15

SK Channel blocker

Answer 15

Apamin

SK2 > SK3 > SK1

Question 16

SK Channels

Answer 16

Calcium-activated K+ channels
Small current ~ 10pS

mAHP <150-200ms - activated following a train of APs
Acts as a rhythmic gate for excitability

3 subunits = SK1, SK2, SK3 - 60% sequence homology

Apamin = blocker
SK2 > SK3 > SK1

SK2 = novel target for cognitive enhancement!

Calmodulin covalently linked to the channel which binds Ca - binding leads to conformational change + channel opening
Calmodulin - tethered to C-terminus domain
2x Ca binding sites = very positive cooperativity

Activated by L-type Ca channels

Native tissue:
SK2/SK1 heteromers with subunits not adjacent to eachother

Important for:
SCN circadian rhythms - daily variation in SK current

Question 17

Blockers of all calcium-activated K+ channels

Answer 17

Charybdotoxin

Question 18

fAHP

Answer 18

<5ms

BK channel activation

Question 19

mAHP

Answer 19

<150-200ms
SK channel activation following a train of APs
Act as a rhythmic brake

Question 20

Tonotopic organisation of chicken cochlea

Answer 20

Partially due to the alternative splicing of BK channels - alters the Ca-sensitivity of the channels

Question 21

SK + LTP

Answer 21

Apamin = SK inhibitor - cognitive enhancer (increase excitability)
BUT weak cognitive enhancer due to the overt side effects at the required dose to enhance - narrow therapeutic window

SK = target for cognitive enhancement

Hippocampus CA1
Because - ACh released when you are aroused
ACh binds mAChR which inhibits SK channels, therefore increased post-synaptic depolarisation, there increase LTP

Question 22

S4

Answer 22

Voltage-sensing domain

Gating particles within S4 ‘move’ in response to a change in Vm = gating current
Non-permissive (resting Vm) –> permissive (depolarised)

Leftward in current + time

Positive arginine residue located every 3rd amino acid - alpha helix - faces the same direction
Mutagenesis studies - 7 arginines = mutate a residue, see a decrease in gating charge

Electrostatic potential map - charge is not evenly distributed along the membrane

• Narrow area of charge = selectivity filter ~12A (membrane = 34A)
• Concentration of electrostatic charge - therefore move a small distance (12), move across the entire membrane field!!!

Current thinking = twists up within S4!

The further the arginine residue up S4, the more influence the arginine residue has on voltage gating
(SK channels - arginine at the bottom of S4!!!)

Question 23

1st cloned K+ channel

Answer 23

Kv1.4 Shaker - A current channel
Mutant lacking channel - shook when in ether

Cloned via positional cloning (chromosome walking)

Question 24

1st crystallised K+ channel

Answer 24

Kv1.2

Question 25

Voltage-gated K+ channels family

Answer 25

``` Most encode for delayed rectifiers 2/3 give rise to channels with A-currents DR/A: Shaker-like Shaw-like Shab-like Shal-like ``` Slo = BK SK 1, 2 + 3 Kv7.2-7.5 = M-current Families divided by homology not function!!!

Question 26

Subunit stoichiometry

Answer 26

Subunit composition = 4 subunits - tetramere MacKinnon Wild-type sensitive to CTX Scorpion toxin Mutant insensitive to CTX Inject wild-type + mutant into xenopus oocytes Produce channels of varying sensitivity due to unique combinations: wwww, wwwM, wwMM, wMMM, MMMM Evidence that Shaker had 4 subunits

Question 27

VGCC Structure

Answer 27

6 TMD P loop - contains tyrosine = TEA binds + blocks channels S4 = voltage-sensing domain GYGD = signature sequence located in the P-loop T1 = tetramerisation domain - tethered arrangement of subunits via their NTD

Question 28

Beta1 subunit

Answer 28

Associate via the T1 domain Mediates N-type inactivation of K+ channels (A current) N-type = ball + chain Ball - cysteine residues = physically occlude the channel - 1 subunit is sufficient! Ischaemia - cysteine residues are oxidised, cannot block the pore: Transient A-current --> inactivating DR Decrease excitability - sensor in oxidative stress! Important in LTP - Aged mice = beta rapidly inactivates K+ channels, delayed repolarisation, AP broadening, increased Ca influx which activates SK + mAHPs = reduced AP spikes - Beta1 knock-out = less Kv inactivation, reduced AP broadening, less Ca influx, less mAHPs, more spikes, more excitable = enhanced learning

Question 29

Beta 2+3

Answer 29

Chaperone subunits - helping the alpha pore subunit reach the membrane Lacking these - alpha retained in the ER! Associates via the T1 domain!

Question 30

Selectivity filter of K+ channel

Answer 30

Crystallised the KcsA selectivity filter Diameter differs due to energy barriers Widest in central cavity Narrowest in selectivity filter (external pore) ~ 12A Internal pore + cavity = inert + hydrophobic; K+ passes through hydrated External pore/selectivity filter (narrow ~ 12A) = lined with polar atoms - K+ gets hydrated Stacks of O2 rings (carbonyl backbone) - forms sites to hold a dehydrated ion 2x K+ in the filter - located at opposite ends of the filter so they do not repel eachother (defined via Fourier analysis)

Question 31

Hill coefficients

Answer 31

< 1 = negative cooperativity 1 = no cooperativity > 1 = positive cooperativity

Question 32

EGTA and BAPTA?

Answer 32

BAPTA = 10x faster - Ca can diffuse up to 10/20nm from the Ca source before BAPTA binds EGTA = Ca can diffuse < 70 nm from the Ca source until EGTA binds Use the 2 chelators to titrate the relevant locations of the channels

Question 33

Signature K+ sequence

Answer 33

GYGD - pore sequence

Question 34

TEA

Answer 34

Blocks tyrosine reside in the P loop - top limb near S6 The expression of K+ with/without tyrosine residue gives rise to K+ channels with intermediate sensitivities to block by TEA Use sensitivity for a marker of how many subunits in a native channel contain the tyrosine subunit

Question 35

Retigabine vs ICA73

Answer 35

Retigabine - binds tryptophan residue in top limb of S5 Need 1x subunit to functional block Physically occludes channel + prevents it from closing ICA73 - stronger; binds S3 and acts on resides in S4 (VSD) Need 4x subunits to bind + functional Fixes the channel in the open state

Question 36

SK channels in vivo

Answer 36

Block of hippocampal mAHPs SK2/SK1 heteromers - Hill slope ~ 1 + IC50 450pM (high compared to SK2 monomers) 1 component to the block (not biphasic) - Hill slope ~ 1 = SK2 subunits are not adjacent to eachother (bimodal distribution; no donating of the EC S3-S4 loop can occur) There is only ONE population of channels Compared IC50 + hill slope

Question 37

Apamin

Answer 37

SK channel blocker - allosteric (Not a 1:1 blockade of the pore - IC50 + Kd would be the same) SK2 > SK3 > SK1 = only toxin which can distinguish subunits! Binds at 2 places: Pore - histidine residue S3 - S4 EC loop - YA amino acids Biphasic blockade (high pos cooperative + high affinity block and no cooperativity + low affinity blockade) + bimodal distribution - apamin requires adjacent subunits for positive cooperativity to occur in order to achieve a high affinity blockade 60% = adjacent, high affinity, positive cooperativity 40% = no adjacent binding subunits, low affinity, negative cooperativity Subunits adjacent - physical communication Donation of the EC S3-S4 loop occurs: -rSK2 (YA --> LV) = cannot bind apamin to EC loop -SK2 (histidine --> asparagine) = cannot bind apamin to inner pore Theoretically - should not get apamin blockade BUT - still get blockade!! The his-->asp donates its 'good' subunit to the adjacent subunit with the 'good pore' - therefore get a functional binding via apamin

Question 38

Why are we interested in SK channel blockade?

Answer 38

SK2 activation - membrane hyperpolarisation via activation of mAHPs (rhythmic gate) SK2 blocker = target for cognitive enhancement, depression, myotonic dystrophy, heart arrhythmias SK1/2/3 - overlapping distriution in the CNS/PNS ***LINK: PD/HD - SK3 inhibitor - myotonic muscular dystrophy ALSO: heart - Repolarisation of cardiac AP mediated by SK - Atrial fibrillation model = Ca leak during AF, enhanced activation of SK channels, accelerated repolarisation + shortened AP duration - SK inhibitor = stops induced atrial fibrillation

Question 39

SK channel enhancer

Answer 39

1-EBIO - anti-convulsant | As dosage increased, mAHP increased + number of mice convulsions decreased