Dr Neil Marrion Flashcards
Delayed rectifier
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
A current
Transient (~10ms) + inactivating due to beta1 subunit
Sets frequency of AP
Activated by AHPs - opposes the progressive depolarisation
Blocked by 4-AP
Where does TEA bind?
Tyrosine in the P loop
M current
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
Retigabine
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)
ICA73
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
Linopiridine
Inhibit M current = cognitive enhancer
Treat vascular dementia + age-related cognitive decline
BK Channels
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
BK + AB42
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
Cole-Moore effect
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
4-AP
Blocks A current
Which current never inactivates?
M current
BK Channel Blocker
Paxilline
A current blocker
4-AP
SK Channel blocker
Apamin
SK2 > SK3 > SK1
SK Channels
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
Blockers of all calcium-activated K+ channels
Charybdotoxin
fAHP
<5ms
BK channel activation
mAHP
<150-200ms
SK channel activation following a train of APs
Act as a rhythmic brake
Tonotopic organisation of chicken cochlea
Partially due to the alternative splicing of BK channels - alters the Ca-sensitivity of the channels
SK + LTP
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
S4
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!!!)
1st cloned K+ channel
Kv1.4 Shaker - A current channel
Mutant lacking channel - shook when in ether
Cloned via positional cloning (chromosome walking)
1st crystallised K+ channel
Kv1.2
Voltage-gated K+ channels family
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!!!
Subunit stoichiometry
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
VGCC Structure
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
Beta1 subunit
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
Beta 2+3
Chaperone subunits - helping the alpha pore subunit reach the membrane
Lacking these - alpha retained in the ER!
Associates via the T1 domain!
Selectivity filter of K+ channel
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)
Hill coefficients
< 1 = negative cooperativity
1 = no cooperativity
> 1 = positive cooperativity
EGTA and BAPTA?
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
Signature K+ sequence
GYGD - pore sequence
TEA
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
Retigabine vs ICA73
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
SK channels in vivo
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
Apamin
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
Why are we interested in SK channel blockade?
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
SK channel enhancer
1-EBIO - anti-convulsant
As dosage increased, mAHP increased + number of mice convulsions decreased
