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Pharmacology of Ion Channels > Dr Neil Marrion > Flashcards

Dr Neil Marrion Flashcards

1
Q

Delayed rectifier

A

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

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2
Q

A current

A

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

Sets frequency of AP

Activated by AHPs - opposes the progressive depolarisation

Blocked by 4-AP

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3
Q

Where does TEA bind?

A

Tyrosine in the P loop

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4
Q

M current

A

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

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5
Q

Retigabine

A

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)

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6
Q

ICA73

A

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

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7
Q

Linopiridine

A

Inhibit M current = cognitive enhancer

Treat vascular dementia + age-related cognitive decline

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8
Q

BK Channels

A 
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

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9
Q

BK + AB42

A

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

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10
Q

Cole-Moore effect

A

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

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11
Q

4-AP

A

Blocks A current

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12
Q

Which current never inactivates?

A

M current

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13
Q

BK Channel Blocker

A

Paxilline

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14
Q

A current blocker

A

4-AP

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15
Q

SK Channel blocker

A

Apamin

SK2 > SK3 > SK1

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16
Q

SK Channels

A

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

17
Q

Blockers of all calcium-activated K+ channels

A

Charybdotoxin

18
Q

fAHP

A

<5ms

BK channel activation

19
Q

mAHP

A

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

20
Q

Tonotopic organisation of chicken cochlea

A

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

21
Q

SK + LTP

A

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

22
Q

S4

A

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!!!)

23
Q

1st cloned K+ channel

A

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

Cloned via positional cloning (chromosome walking)

24
Q

1st crystallised K+ channel

A

Kv1.2

25
Q

Voltage-gated K+ channels family

A 
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!!!

26
Q

Subunit stoichiometry

A

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

27
Q

VGCC Structure

A

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

28
Q

Beta1 subunit

A

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
29
Q

Beta 2+3

A

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

Associates via the T1 domain!

30
Q

Selectivity filter of K+ channel

A

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)

31
Q

Hill coefficients

A

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

32
Q

EGTA and BAPTA?

A

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

33
Q

Signature K+ sequence

A

GYGD - pore sequence

34
Q

TEA

A

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

35
Q

Retigabine vs ICA73

A

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

36
Q

SK channels in vivo

A

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

37
Q

Apamin

A

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

38
Q

Why are we interested in SK channel blockade?

A

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
39
Q

SK channel enhancer

A

1-EBIO - anti-convulsant

As dosage increased, mAHP increased + number of mice convulsions decreased

Pharmacology of Ion Channels (9 decks)

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