Membrane excitability Flashcards
What is Eion?
Membrane potential at equilibrium
K+ -89mV
Na+ 66.5mV
Mg2+ 8.4mV
Ca2+ 140mV
Cl- -89.9mV
Which phospholipids are involved in membrane structure?
Phosphatidylethanolamine (PE)
Phosphatidylserine (PS)
Phosphatidylcholine (PC)
Phosphatidylinositol (PI)
^all have head group w/ glycerol backbone + 2 fatty acids
Sphingomyelin - choline head w/ sphingosine 18C backbone + 2nd fatty acid tail
forms very low permability barrier to water (polar) + impermeable to ions (charged)
What glycolipids are involved in membrane structure?
Galactocerebroside - galactose w/ sphingosine + 2nd fatty acid tail
Gangliosides
Cholesterol
Rigid steroid ring structure w/ polar -OH group + hydrocarbon tail
Increases rigidity/stability + redcues permeability, anti-freeze, can flip between leaflets easily
What determines membrane fluidity?
Double cis bonds increase fluidity in unsaturated tails - kinks stop lipids packing close together compared to saturated
Longer C chains -> less fluid
- determines membrane thickness/width
Describe lipid composition in membranes
50 lipid mols to 1 protein, but proteins 30-45% mass
Outer leaflet - PC, SM, glycoplipids (cell contact)
Inner leaflet - PE, PI, PS (signalling via cytoplasm)
- distribution is asymmetric (transverse diffusion)
Which molecules regulate transverse diffusion in the membrane?
Flippases - outer to inner leaflet e.g. P4 type ATPase, P1 type is Na+/K+ ATPase, moves PS/PE
Floppases - inner to outer leaflet e.g. ABC ATPase from ABC transporter superfamily
What happens if lipid asymmetry is lost?
Acts as signal for cell fate/state
PS in outer leaflet -> signal phagocytosis
Inhibition of flippases + activation of Ca2+ dependent phospholipid scramblase -> more PS in outer leaflet
Key step in apoptosis + aslo seen in oxidative stress, platelet plug formation in clotting, immune defence against virus, sickle cell disease
Integral transmembrane protein structure
Polar a.acids found between inner protein + aq env., they prefer polar environment
Non-polar are hydrophobic so embedded within protiein structure
Synthesised via ribosome-translocon (N or C terminal insertion possible) - when alternated sequentially, multipass proteins generated
List 3 ways proteins can be intracellularly anchored to the membrane
N -myristolation at N-terminal glycine - anchors proteins w/ 14C myristoyl chain, needs N terminal Met residue adjacent to Gly (PKA catalyric subunit, Protein Phosphatase Calcineurin PP2B)
S-palmitoylation at cysteine - anchors proteins w/ a 16C f.acid palimtoyl group at any point in peptide sequence (GAD, t-SNARE, SNAP25)
S-Isoprenylation at C-terminal cysteine - anchors w/ isoprenyl group (5C + 1 methyl side group)
(GTPases like Ras & Rab)
many of these modifications allow reversible binding - subject to conformational change
How are proteins anchored to membrane extracellularly?
Gllycolipid - glycosylphosphatidylinositol (GPI) added to protein C terminal by covalent peptide bond
Phospholipases can relase proteins as part of signal pathway
(AChE, neural cell adhesion mol N-CAM, ephrin-A ligands)
How do proteins move in membranes?
Can spin about z axis, can change conformation, can move laterally.
BUT do not translocate transversely or rotate (flip)
How is lateral diffusion controlled?
Lipid rafts - tightly packed areas of high cholesterol, SM + saturated f.acids, low PC)
-> allow lateral segregation
More organised microdomains so can compatrmentalise membrane processes to discrete locations
- associated w/ caveolae formation (invagination)
Basic ion channel structure
a-subunits form channel:
- as tetramer (24 in Kir, 64 in VG K+, 74 inBK K+)
- as monomer (24*1 in Ca2+ & Na+)
In Na+ a-subunit is channel forming pseudo-tetramer
P loop present in pore region + determines selectivity
What is a gating mechanism?
Twisting, titling + bending of subunits/TM a-helices to an open or closed conformation.
Describe 4 mechanisms of generalised gating
Ligand binding to fast NT receptors e.g. glutamate OR intrecallularly by ATP, cAMP, cGMP to By-subunit sof G proteins
Change in membrane voltage detected by charged TM4 (Voltage sensor) - positions shifted by hyper/depolarisation
Intracellular phosphorylation/dephosphorylation - increases oepning of leaky K+ channels + gap junctions
Mechanical distortion - plas mem mechanosenstive, increased opening directly or via cytoskeleton
e.g. sensory transduction in cochlea + TRP channels in skin
Describe the types of inactivation
Localised (C-type) - region in pore wall alters conformation occluding pore OR slectivity filter changes conformation reducing ion transfer - short amino acid sequence
Particle (N-type) - free intracellular region plug the pore (ball and chain gating) - long amino acid sequence or subunit
What are ion channels selective for?
Charge sign
Charge density (ion size, radius, charge size)
K+ channels have 100 fold selctivity for K+ over Na+, a = 0.01
Why does RMP vary across different tissues?
In skeletal muscle + axons, close to -90mV (Ek) -> suggests single class K+ channels w/ high degree selectivity
Plasma mem in both contains Kir + K2p channels -> have TVGYG sequence (1 a.acid per subunit forming selectivity filter
Number of channels does not affect RMP, but relative number of channels with different selectivites does
What are the different K+ channel selectivity filters?
A. acids in P loop normally highly conserved, but some altered from TVGYG seq.
- HCN, hyperpolarisatio activated nucleotide gated channels (neuronal + cardiac tissues)
- CNG, cyclic nucleotide gated channels (retina photoreceptors)
Both have lower K+/Na+ selectivity at 4 fold a = 0.25
Less selective w/ RMP at -20mV
What is Iion?
Difference between TM voltage Vm and Eion
- indicates ion influx/efflux
Iion = Vm - Eion
What is the conductance of a membrane?
Ion channel permeability, opposite of resistance.
Variable -> activation/inactivation
Determines size of Iion at particular Vm relative to Eion.
Vm - Eion = Iion/Gion
zero current flow when Vm = Eion
Capacitance across a membrane
Phospholipid bilayer acts as non-conducting capacitor.
Extra + intracellular fluids are conudctors - membrane dielectric so maintains chareg separation.
Membrane has capacative currents (Ic) that flow when mem potential + charge changes
- currents dont affect ion concs
- Specific membrane capacitance is 1µFcm-2 (biological constant)
SA can be used to detrmine total capacitance
What is a passive response in a membrane?
Voltage responses lag behind membrane current.
- negative current (outward) causes hyperpolarisation
- when current flows through membrane, Vm not = RMP
time constant helps determine how much V response lags behind current - directly proprtional to capacitance but inversely to conductance
Impact of capacitance on membrane
Usually constant as cell SA does not change - determines frequency response
If duration of stimulus short, then intensity must be high.
But if duration is long, then lower intensity can still trigger an AP.
Impact of conductance on membrane
Does not change + has 2 effects:
1) determines where steady state voltage level of excitable membrane will be, hence whether potential reaches threshold
2) affects time constant t = C/G, increased C means more current needed but it reaches threshold quicker
- neurontransmission: large C changes speed up responsiveness
both effects interact
How were voltage clamps first used to measure membrane/channel function?
1st H+H described Na+ & K+ in giant squid axon 1950s -> coordiantes water ejection from mantle cavity in escape response
H+H - Hodgkin & Huxley
Voltage clamp mechanism
Amplifier monitors cell mem voltage + miantains it w/ potential injections (negative feedback mechanism)
Purpose to measure current flow of ions + determine conductance using Ohms law.
Current that miantains set voltage level must equal that generated across membrane causing change in potential.
Giant axon current mechanics
Unexpected hyperpolarisation triggered AP - very small capacitive transient current (inwards) to obey capacitance laws
Expected depolarisation triggered AP - transient inward followed by sustained outward current (summation both K+ & Na+)
In one experiment H+H used ion substitution - choline+ used instead of Na+ -> early inward current disappeared
Whole cell patch clamp
reliably generates macroscopic current
I = G (Vm - E rev)
Erev is potential where macrosopic current reverses direction
- many channels contribute
- little variation between trials
- onset + duratio constant
- accurate est of whole cell conductance changes over time
Current flow relatively large 0.01-10nA, whole cell conductance 0.1-100nS.
Vm - E rev is driving force
G is whole cell conductance
Single channel patch clamps
Can be outside-out or inside-out config.
i = y (Vm - Erev)
Erev is potential at which microscopic current reverse direction, if channel highly ion selective Erev = Eion.
- timing + duration of opening varies massively (imprecise - stochastic)
- may not open at all
- single channels unreliable
current flow small <0.01nA, single channel conductance <0.1nS
y is single channel conductance
Vm - Erev is driving force
What do N and F respresent when linking whole cell and ion channel properties?
N - total number of channels
F - fraction open at any time (0-1)
total macroscopic mem current I = N* F * i
total macroscopic conductance G = N * F * y
i = sum of microscopic currents
What 2 methods can be used to conisder conductance?
1) Ohms law
2) Slope of line:
- chord conductance is line on I-V plot from any point to Erev
- slope conductance is between any 2 points
If system is ohmic: chord = slope (linear)
conductance indicative of open channel number
Membrane permeability to different molecules
Lipids + gases -> very high
water -> modest
glucose + a.acids -> low
ions -> very low/none
Meyer-Overton rule - membrane permeability of a substance is directly proprtional to its solubility in oil
-> used by pharma to predict drug penetration
lipid permeability increased at higher pH -> e.g. cocaine chewed w/ alkaline
How is transport via membrane proteins different to simple diffusion?
Saturable process - according to M-M kinetics
Has Jmax & Km
Ion pump characteristics
Transport 1 or more substrate (1 against a gradient)
- enzymatic activity (ATPase) for primary AT
e.g. Na+/K+ pump 3Na+ out 2K+ in
Types of ion pumps
P-type: Na+, K+, ATPase, flippase
F-type: ATP synthesis in mt
V-type: H+ pump in organelles
ATP binding cassette (ABC) proteins:
- P-glycoprotein (MDR1) -> drug resistance
- CFTR
- Sulphonylurea receptor
What type of proteins come under the solute carriers (SLC superfamily)?
Transport 1 or more substrate (at least 1 down gradient)
Very diverse substrates.
66 canonical sub-families + 5 non-canonical sub-families
- co-transporters/symporters (2 or more in same direction)
- exchangers/antiporters (2 or more in oppsosite directions)
- f. diffusion proteins -> GLUTS for glucose, GLUT4 is for insulin dependent glucose transport
Examples of cotransporters
SGLT1 for Na+/glucose (2Na+ & 1 glucose in)
NKCC1 for Na+, 2Cl- & K+ in -> excitation by eGABA in suprachiasmatic nucleus (circadian master clock), secondary AT (no ATP directly used)
Examples of exchangers
NHE for Na+/H+ exchange (pH)
AE (anion exchanger) for Cl-/HCO3- exchange
SLCs as drug targets
Amphetamines -> NET (SLC6A2) & DAT (SLC6A3 for dopamine)
SSRIs - inhibitors of Na+/serotonin co-transporter (SLC6A4)
New: SGLT2 inhibitors for diabetes
SSRI - Selective serotonin reuptake inhibitors
What do membranes use to enhance water membrane permeability?
Aquaporins - e.g. AQP2 insertion by vasopressin in kideny collecting duct increases water permeability
Discovered by AGre 1992 - worked on rhesus proteins in RBCs
Family of 9 proteins: AQP4 is main CNS channel
- knock out in mice showed improved outcomes for stroke (less swelling) , reduces inflammation
What is the fundamental role of an ion gradient across a cell membrane?
Maintain cell volume - pre dates + gives rise to membrane potential
Transport influenced by both chemical gradient & membrane potential.
Nernst equation function
Convert chemical gradient to an electrical gradient
EC gradient = Vm - Ex
Ex is Eq for ion x.
What is the ‘pump leak’ hypothesis?
1950s - K+ efflux mainatined RMP after import in via ATPase
BUT not enough, 1980s discovered Cl- has major role in maintaining cell vol.
Cell volume = equilibirum between extruders v accumulators
Describe the regulatory volume increase (RVI) mechanism
Net gain on K+ & Cl- using NKCC1, very slow.
Driven by Na+ gradient, Na+ removed by ATPase.
Water movement in via osmosis.
Describe the regulatory volume decrease (RVD) mechanism
Net loss of K+ & Cl- using KCC & respective ion channels.
Driven by K+/Cl- gradient
Use either co-trasnprter or separate ion channels.
In atypical cells (mature neurons, pancreatic a-cells, skeletal muscle): no Cl- gradient so actively extrude Cl-
How is Cl- concentration determined in cells?
Ratio of NKCC1:KCC2
notion of low Cl- in all cells is incorrect - Cl- accumulates in most cells, then efflux causing depolarisation
e.g. high Cl- in hippocampus, suprachiasmatic nucleus, substantia negra
Example of cell regulating osmolarity during cell migration
decreased cell vol in glioma cells (loss of K+/Cl-) allowing them to squeeze between CNS cells during metastasis
- chlorotoxin (Cl- channel blocker) inhibit glioma cell migration in vitro
- in vivo fluorescently labelled chlorotoxin can be used to identify tumour cells -> surgery
Importance + regulation of pH
All eznyme have optimum pH - denatured if skewed.
Extracellular pH (~7.4) regulated by lung + kidney (HCO3-/CO2 buffering)
Intracellular pH (~7.2) regulated by transport protein combinations
pH = -log10 [H+], optimum [H+] = 20-100nM
How can pHi be altered?
Metabolism - production of acidic metabolites
Ischaemia - decreased blood flow increases anaerobic metabolism, ASICS stimulated
Membrane transprot proteins e.g. Ca2+ ATPase (PMCA) or HCO3= permeable channels
-ve Vm cuases electrochemical gradient for H+ influx
ASIC - acid sensing ion channel
How is pHi measured?
Tsien developed:
- ph sensitive fluorescent inidcators (BCEF, SNARF)
- Ca2+ sensitive fluorescent inidcators (FURA2-AM)
Laser uses monochromatic lght to excite cells -> emit at different wavelengths depending on [H+]
can measure at euilibirum or when equilibrium is disturbed -> functional studies
e.g. in cardiac myocytes, dye attached to lipid permeable ester so it can diffuse into cell - then hydrolysed so trapped in cytoplasm
How is NH4+ used to determine [H+]?
Acidification of cells - ammonium/alkaline added, H+ influx as a rebound action but will then equilibrate if left alone
Influx/extrusion can be determined using EIPA ( NHE inhibitor)
- NHE is the Na+/H+ exchanger
If NHE blocked then [H+] remains high
Give examples of acid extuders and loaders
Extruders - NHE, NBC (SLC4 family)
Loaders - CHE (SLC26, OH- out), CBE (AE or SLC26, nHCO3- out)
Activity of both is lowest in the permissive pH range - stable pH at ~7.2
NHE - Na+/H+ exchanger
NBC - Na+/HCO3 co-transporter
CHE - Cl/OH exchanger
CBE - Cl/HCO3 exchanger
Why and how is [Ca2+] kept very low?
- It has high affinity for phopshate ions -> insoluble precipitate, calcification
- key for intracellular signalling, mjst remain low until use
1) extrusion across cell mem - NCX expels Ca2+, PMCA actively transports Ca2+ out
2) Sequestration into organelles - SERCA exchanges Ca2+ for H+ in ER/SR
Electroneutral vs electrogenic extrusion
electroneutral - non et movement of charge e.g. NHE, NKCC1
electrogenic - net movement of charge generating small electrical current e.g. SERCA, SGLT1/SLC5A1
Which receptors allow Ca2+ entry and release from cell/organelles?
Cell: VGCCs, NMDA-Rs, store operated
Organelles: IP3 GPCRs, ryanodine
Extrusion + sequestration effective off switches
How can the same signal induce a different response?
Ca2+ is universal messenger -> physiology
Temporal differences -> variation of Ca2+ oscillation frequency between ACh (faster) and CCK NTs
Spatial differences -> Ca2+ waves in isolated cardiac myocytes (imaging), FURA2-AM changes 380 to 340nm when Ca2+ present
- GCamP6 is genetically encoded calcium indicator, measures Ca2+ in real time to study mice behaviour/brain acitivity
List the different types of anatomical barriers
Inert - meninges around brain
Physiological - skin, intestines, airways, reproductive tract, kidneys, liver, exocrine glands
Specialised - blood-brain, blood-CSF, blood-retina, blood-testes, placenta
Physiological + specialised are active (except skin) -> protective mechanism
other CNS barriers inc retinal + blood-spinal chord
How can the NS influence secretion?
Increased IP3 via Gq increases [Ca2+]
Loss of K & Cl decreases cell volume
HCO3- efflux increases [H+]
How can transport occur in epithelial cells?
Linked by junctional complexes (claudins + occludins)
Claudins determine tightness + selectivity of junctions (paracellular transport)
Transcellular transprot requires polarity.
What are the 3 main interfaces in brain protecting neurons from blood-bornes substances?
BBB (cerebral endothelium) - tightly regulated, discovered by Ehrlich, Evans (blue dye IV into rat), Goldmann (dye into CSF)
Blood-CSF interface - epithelial cells of choroid plexus
CSF-blood interface (avascular arachnoid epithelium lies under dura + encases brain)
Cerebrospinal fluid (CSF)- transport of substances to the brain + drainage of waste products/toxins
Blood brain barrier structure
From cerebral endothelial cells - polarized (apical transporters), inactivates toxic substances + removes them.
Connected by tight + adherens junctions (characterised by Reese+ Karnovsky)
- continuous actin cytoskeleton has high electrical resistance (retention of ions)
BBB is absent from circumventtricular organs (CVO)
e.g. pineal gland, lamina terminalis, neurohypophysis, area postrema
-> these are fenestrated (leaky) e.g. for hormone release.
- specialised cells -> tanycytes & ependymal cells allow substance diffusion into CVO but not beyond.
How are different diseases associated with blood brain barrier (BBB)?
Chronic - MS, autoimmune encephalomyelitis, AD
Acute - ischaemic stroke, hypertension, seizure
-> associated w/ misregulation of tight junctions or pumps/carriers
Blood-CSF barrier structure
CSF formed in 3rd + 4th lateral ventricles by choroid plexus (CP)
- same comp as interstitial fluid (ISF)
Provides nutrients + signalling conduits for brain + spinal fluid.
CP capillaries are fenestrated.
Tight jnctions of ependymal cells of CPs form blood-CSF barrier.
Structure and function of ependymal cells in choroid plexus (CP)
Epithelial like:
- ionic pumps on apical surface (Na+/K+ ATPase) -> chemisomotic energy for fluid formation by CP
- water follows osmotic gradient set up by 3Na+ removal (CSF formation helped by blood flow)
Role of astrocytes in BBB function
Barrier properties within brain parenchyma via upregulating tight junction proteins.
- large no. of K+ channels that can activate/inactuvate them
Astrocyte-endothelial interact goes both ways -> endothelium signals astrocytes e.g. AQP-4 in astrocytc feet around vessels in brain parenchyma
Reese + Karnovky contend barrier function at endothelia level not astrocytes
What are the protective functions of barriers for the brain?
Control immunologic status of brain- no pathgen entry.
Tight junctions do not allow ions to move passively into brain -> prevents fluctuations
Proteins (albunim) + blood cells prevented from passinginto brain -> could damage neurons + interfere w/ osmotic homeostasis
Why cant ivermectin be used in collie breeds?
Mutation reduces expression of p-glycoprotein.
Ivermectin is anthelmintic drug to treat parasitic worms - lipid soluble so corsses BBB then removed by p-glycoprotein.
-> accumulates + neurotoxic if not removed
How can different ion channels be classified?
Electrophysiological properties - selectivity, voltage-dependence, current kinetics
Pharmacology - agonist/antagonist activity of specific drugs
Modulation by regulatory mols - Ca2+, G protein, ATP
Structure - pore forming subunits, auxiliary subunits, a. acid sequence
genetic diversity underlies ion channel diversity - subtypes expressed according to cells requirements
Diversity in K+ channel a subunits
- calcium activated
- inwardly rectifying
- two pore domain
- voltage gated
Different types of Ca2+ VG channels
L type: 1.1-1.4, high voltage activated, DHP sensitive
P/Q type 2.1 & N type 2.2 & R type 2.3, high voltage activated, DHP insensitive
T type: 3.1-3.3, low voltage activated
L- type -> muscle contraction + secretion
P/Q type -> CNS transmitter release
N type -> PNS & CNS transmitter release
R type -> transmitter release
T type -> cell excitability e.g. pacemaker activity in heart
What is the minimal structural requirement of a pore forming subunit of the VG superfamily?
All related.
S5-P-S6 motif in pore lining.
- some have charged a. acids -> voltage sensors
Structure of VG K+ channels
6 a-helical segments
4x a subunits form functional channel + 4x B subunits (increases trafficking, surface expression + modulate gating)
Voltage sensor in S4 (+vely charged w/ R or K every 3/4 residues) , S5-P-S6 contains selectivity filter
What is electromechanical coupling?
Occurs when any VG channel opens - charged a.acids move through mem electrical field, coupling electrical work to opening process
GTGC is gating charge transfer centre (-ve charged), S1-S3 in VG K+
How are VG K+ channels activated/inactivated?
Activation: S4 moves via pariwise interactions w/ -ve GCTC, displaces S4-S5 linker -> opening
Inactivation: opening immediately triggers or N- or C- type inactivation (ball and chain)
*C type is slower
Function of state dependent blockers
State dependnece relies of voltage dependence.
Local anaesthetics preferentially bind channel states (lidocaine, prilocaine) - use dependent block to bind I, III & IV of S6 to lock channel in inactive state
Structure of VG Na+ channels
Pore forming a-subunit has 4 copies of pseudosubunits (Kv like structure).
4B accessory subunits -> modulate gating, regulate surface expression + function as adhesion mols.
Pore forming unit highly conserved so difficult to target specific channels.
- essential for AP propagation so targets for various drugs
encoded by same gene family
Use of TTx to classify different VG Na+ a-subunits
Based on sensitivity + resistance.
TTx is a toxin that binds extracellular region of S5-S6 loop (TMQDxWE)
If sensitive (TTx-s), then x is aromatic (Y or F), causes larger Na+ influx, rapidly inactivates
If resistant (TTx-r), x is polar (C or S), smaller amplitude, slower inactivation
VG Na+ chanelopathies
1.1-2: Lof .1 and GoF .2 cause epilepsy
1.5 mutations: cardiac arrhythmias (LQT syndrome)
1.7:
- GoF causes erythromelalgia (IEM), paroxysmal extreme pain disorder
- LoF causes congenital insensitivity to pain (CIP)
VG Ca2+ channel properties
Very similar to Na+, has inverted bell shape I-V relationship.
EC gradient inwardly directed, but opens later (-30mV) than Na+ (-50mV), in response to larger stimuli vs Na+ which sets threshold (AP firing)
Raises intracellular Ca2+, important 2nd messenger.
CaVs convert elctrical signal directly to biochemical via [Ca2+] regulation.
Features of low vs high voltage activated Ca2+ channels
Low - activated by small depolarisations, stimuli close to RMP, regulate excitability.
Peak current at -30mV, opens at AP threshold. No B subunit
High - activated during AP, trigger NT release in nerve terminals
Peak current at 0-10mV. 1:1:1 subunit stoichiometry.
L type are dihydropyridine sensitve vs other are not -> early classification
Structure of VG Ca2+ channels
Very similar to Na+, 4 pseudosubunits.
a1: 10 subtypes, single polypeptide chain w/ 4 TM domains, sensor in S4, pore formig loop in S5, joined by intracellular loop
B: 4 subtypes, B1 has laternative splicing -> diversity, binds intracellular 1-2 loop
a2delta: auxiliary subunit, TM protein w/ large extracellular head -> interacts w/ channels + preteins
- targeted by gabapentinoids that disrupts its trafficking (anticonvulsants for chronic pain)
L type Ca2+ antagonists
1,4-dihydropyridine (DHPs) e.g. nifedipine (anti-hypertensive), smooth muscle favourable
Phenylakylamines (PAAs) e.g. verapamil (anti-arrhythmic), cardiac favourable
Benzothiezapines (BZTs) e.g. diltiazem, anti-arhythmic/hypertensive), intermediate
^all target CaV 1.2 a1 subunit + lock it in inactive state (gating
N type Ca2+ antagonist
Ziconotide (synthetic) blcoks channel + reduces NT release for chronic pain.
List the different types of K+ channels
Kv - delayed rectifiers + transient A type current
Kca - small (IK and SK) and large (BK) conductance
Kir - inward rectifiers
K2P - two tandem pore domain
Role instabilising RMP, -70mV largely reliant on Ek (-89mV).
Why is Kv tetramer formation important?
Allows channels to heteromultimerise -> diversity
Differ from parental channels in biophysicial/pharmacological properties.
e.g Kv 2.1/9.3 channels have double conductance of Kv2.1 (greater efflux)
Hetermoeric channels activated by smaller depolarisations (more powerful)
Role & function of delayed rectifier channels (Kv)
Control of AP duration
- short if current activates fast (nerves/skl muscle)
- long if current actuvates slow (heart)
Pharma - blocked by quaternary ammonium ions (TEA)
Role & function of transient A type current (Kv)
Controls AP interspike interval
- short if current small
- long if current large
Pharma - blocked by 4-AP (aminopyridine) -> dendrotoxin
Role & function of BK (Kca)
Big conductance
7TM a-helices instead of 6 in VG Kv.
Sensitive to both voltage + [Ca2+]i
- Ca2+ binds CTD ‘calcium bowl’ on S6
Works concurrently to increase K+ efflux & repolarise membrane.
Functionally couple to N- type Ca2+ channels, mediate fast hyperpolarisation in neurons to inhibit NT release.
Pharma - blocked by charybdotoxin, iberiotoxin + TEA
Role & function of SKca & IKca
Small conductance
Voltage insensitive, activated by low [Ca2+]i, < 1.0uM.
6TM, no +ve charged residues in S4
- S6 binds Ca2+ via calmodulin (indirectly) at C-terminal end
Causes AP repolariation + hyperpolarisation in neurons + muscle, regulates AP firing frequency.
Pharma - SK blcoked by apamin, IK blcoked by charybdotoxin + TEA
Roel & function of Kirs
Inward rectifiers
Illicit inward K+ current under artificial extreme -ve conditions.
Outward current less expected due to blocking by intracellular Mg2+ & polyamines
Only have 2TM domains -> S5-P-S6 equivalent
Vm rarely < Ek SO
- strong inward rectifiers needed to stabilise RMP by conducting K+ at potentials close to RMP
- weak rectifiers pass more outward current at depolarised Vm to mediate K+ efflux & rapid Vm repolarisation
Difference between strong & weak Kirs
Vm rarely < Ek SO
- strong inward rectifiers needed to stabilise RMP by conducting K+ at potentials close to RMP, close when VM strongly depolarised
- weak rectifiers pass more outward current at depolarised Vm to mediate K+ efflux & rapid Vm repolarisation
Kir examples with different functions
GIRKs & KATPs
Kir 3 (GPCR reg, GIRK) - mediates inhibitory effects of many GPCRs, antagonist is ifenprodil
Kir 6 (ATP sensitive, KATP) - close when ATP is high, promotes insulin secretion, inhibitors are glibenclamide & tolbutamide
What are 3 atypical excitable tissues?
Heart:
Sa node - pacemaker activity in HCN
Cardiac myocytes - ventricular AP
Pancreas:
Insulin secreting B-cells - sensing glucose (Kir 6.2)
3 phase cycle of SA node pacemaker potential
0: upstroke - L type Ca2+ current, slow depolarisation
3: repolarisation - K current from Kir 3.1-.4 & hERG
4: pacemaker - If is pacemaker current (diastole), It is transient sub-threshold Ca2+ current 3.1 -> slow rising ramp-like potential
hERG - human Ether-a-go-go related gene Kv 11.1
Same in Av node
What is the pacemaker current?
If - funny current
Inward, but activated by hyperpolarisation threshold -50 to -40mV
Different to conventional V- dependent currents.
Eion -20 to -10 mV (mixed Na+ & K+ permeability)
- gradually depolarizing the membrane potential preparing for L-type Ca2+ opening
What is If pacemaker current caused by?
HCNs
CNBD - cyclic nucleotide binding domain shifts voltage dependence of activation to more depolarised level, currents generated faster
-> similar topology to K channelsw/ modified ion selectivity
Hyperpolarization-activated cyclic nucleotide-gated (HCN)
Parasympathetic autonomic modulation of If
Parasymp input -> vagus nerve decreases HR (bradycardia),
ACh release from the parasympathetic nerve activates M2 muscarinic receptors.
This decreases cAMP levels (Gi), leading to reduced PKA activity.
HCN channels become less active, reducing the inward Na⁺ and K⁺ current (I_f), which slows down the depolarization of pacemaker cells.
This results in a slower heart rate.
Symptathetic autonomic modulation of If
Thoracic 1-4 spinal nerves increase HR (tachycardia)
- noradrenaline (NA) increases If, speeds up pacemaker firing
- mimicked by isoprenaline B AR-agonist via B1-adrenergc receptors (Gs increases cAMP which increases HCN Na+ & K+ conductance)
Ivabradine function
HCN blocker slows HR
-> for heart failure w/ elevated HR, chronic stable angina (insufficient myocardial perfusion)
Reliability of release + AP at the NMJ
1:1 between each motor neurone input + associated muscle fibre.
- substantial EPP from single motor neurone (suprathreshold up to 40mV)
- ~1000 active zones in single endplate, each vesicle generates mEPP ~ 0.4 mV -> quantal release
- motor AP generated after fusion of 100-200 primed vesicles
5 phase cycle of ventricular myocyte AP
0: upstroke, fast Nav1.5 activation
1: initial rapid depolarisation, Nav1.5 inactivated + transient Kv4.2/3
2: plateau phase , L type Cav1.2, slow Ik by Kv7.1
3: Ik slow (Kv7.1) & Ik rapid (Kv11.1 - hERG), Ik Kir2.1-3
4: resting Ik Kir2.1-3
hERG role + function
Highly selective + important for extended plateau in cardiac AP
K+ channels w/ unusual features:
- typical activation > -40mV
- partial inactivation when potential >0mV
- rapid reversal on hyperpolarisation -> increases current (paradoxical resurgent current, Kr), appears swtiched off but reappaears at very low potentials
- timing of repolarisation & QT interval length
hERG - human Ether-a-go-go related gene Kv 11./ Kr
Ventricular fibrillation and LQTS
LQTS - Ling QT syndrome
Prolonged AP -> early after-depolarisation + run of spntaneous activity
Vent fib causes arrhythmias/tachycardia, loss of consciousness & sudden death
LQTS - abnormally long interval between onset of excitation/contraction + their relaxation (depolarisation + repolarisation)
Cardiac side effects/toxicology (methadone, erythromycin, halodiperol)
- drugs need to be tested for hERG blocking action
B cells response to glucose in islets of Langerhans
Stimulates uptake, metabolism + storage.
- glucose transported in via GLUT2 + phos trapping it inside cell
- mt produce ATP
- ATP sensitive K+ channels contirbute to RMP, increase in ATP:ADP ratio -> channels close
- membrane depolarises as other channels contribute to RMP
- VG-Cav open allowing Ca2+ influx
- Increase in [Ca2+]i triggers secretion of insulin via exocytosis
ATP-sensitive K+ channel acts as ‘glucose sensor’ - found in other excitable cells
SUR/Kir 6.2 channel
ATP sensitive K+ channel
Kir combines w/ 4 SUR subunits
SUR1 is 4 regulatory sulfonylurea receptor (SUR) subunits -> SUR1, an ATP-binding cassette protein (ABC)
If ADP binds SUR1 - open more
If ATP binds Kir6.2 - closed more
sensitive to ATP:ADP
forms K atp
Channelopathies in SUR/Kir6.2
LoF - SUR/Kir6.2 closed -> B cell mem depolarisation, increased insulin secretion
e.g. congenital hyperinsulinism (CHI)
GoF - remain open as Kir6.2 less sensitive to ATP/ADP ratio -> B cell mem maintains hyperpolarisation, decreased insulin production
e.g. neonatal diabetes, predisposition type 2 diabetes
LoF allows Ca2+ influx but GoF does not
Key features + function of chemical synapse
- mols stored in vesicles + diffuse across a gap (cleft)
- relatively slow (0.5msec)
- unidirectional
- majority of transmission in NS
Allows modification of NS:
- neural composition -> integrates many inputs
- plasticity for memory + learning
- targets for drug action (broad range for target flexibility)
Definition of a neurotransmitter
Synthesised + stored in pre-synapti neurone, released upon stimulation in depolarisation/Ca2+ dependent manner
- physiological effects at post synaptic mem
- transmitter recognition + signal transduction
- transmitter removal
Types of NT
Small mols:
Amino acids - gly, GABA, glu
Amines - NA, dopamine, serotonin, ACh
Purines - ATP, adenosine
Peptides- endorphins, neuropeptide Y, somatostatin, substance P
Dale’s principle
Old idea that neurones release just 1 NT at its synapses
Challenged by:
- small mol + protein release by interneurones (GABA + enkephalins at striatum)
- many small mol transmitters in some pathways (L glutamate & dopamine)
Key features + function of an electrical synapse
Ions, 2nd messengers or metabolites can pass through gap junctions/connexons linking adjoining cell membranes.
- very fast signalling
- bidirectional
- direct electrical coupling between cells e.g. heart
- rare in NS, inhibitory interneurones or local networks e.g. neocortex & retina
SSVs vs LDCVs
SSV - small synaptic vesicle
LDCV - large dense cored vesicle
- SSV 40nm diameter vs 100nm
- SSVs in synaptic active zones vs LDCVs all over
- SSVs close to VG-Ca2+ channels
- SSVs use small NTs, LDCVs use peptides (+NA)
- need 200uM [Ca2+] for release, 5-10um in LDCVs
- single AP vs repetitive in LDCVs
- constitutive biogenesis (recycling) vs LDCV regulated (ER derived so no recycling)
Vesicles derived from ER + trafficked to docking sites.
Evidence for full fusion/collapse of synapse
Heuser + Reese - slam freezing technique
- rapidly cooling frog NMJ on metal block after stimulation of axon fibres by ACh
- freeze fracture EM -> clear pits formed
But activity led to increased SA -> vesicles recycled to endosome (clathrin coated)
Alternative model for NT release into synaptic cleft - kiss & run
Full fusion not required
- NT leaks out of small fusion pores
- SSVs recycled intact from cell mem + not via clathrin coating via endosome
Kiss & run mechanism vs classical
K&R:
- fast recycling
- low capacity
- favoured at low frequency
- flickering capacitance (brief vesicle interaction w/ mem)
Classical:
- slow recycling
- high capacity, many vesicles over time
- favoured at high frequency stimulation
- increasing capactiance steps (full membrane collapse)
Steps in classical vesicle cycle
- Docking- vesicle docks at active zone (differ by vesicle no. between neurones)
- Priming - vesicle maturation + made competent, requires ATP, protein conf changes drives release
- Fusion/exocytosis - full fusion of mems requires Ca2+ & involves Ca2+ sensor protein, NT discharged ~1msec
- Endocytosis - recovery of fused membrane, recycling triggered by intracellualr Ca2+, skeletal lattice formation (clathrin monomers), ~5 secs + ATP dependent
- Recycling - conserves vesicle mem via endosome, refill w/ NT -> ATP-dependent
DPFER
Release machinery proteins
Snare proteins:
Vesicle associated - synaptobrevin (VAMP) & synaptotagmins (v-SNARE)
Plasma mem associated - SNAP-25, syntaxins (t-SNARE)
Munc18s - ensures priming of vesicles
NSF - allows vesicle to disengage from mem
Docking machinery in action
Synaptobrevin, syntaxin, SNAP-25 make 1:1:1 trimeric complex
-> coiled coil quaternary structure of a-helices from SNAP-25
Priming release machinery in action
Snares form tighter complex -> ‘zippering’ forms SNARE-pins
- ATP-dependent
- assisted by Munc18s, binds to syntaxin Habc domains
Fusion release machinery in action
Synaptotagmin is Ca2+ sensor (65kDa) found on vesicles
- binds SNARE-pins in absence of Ca2+
- Binds phospholipids in presence of Ca2+ (cia C-terminal)
Ca2+ binding can cause synaptotagmin to pull vesicle into membrane
How can reliability of release be defined?
Pr = mean no. vesicles in release (m) / number of active sites (n)
e.g. for NMJ, Pr = 100-200 /1000 = 10-20%
-> microscopic ACh release not very reliable
Pr is low, fusion failure rate is high
Why is the NMJ over-engineered?
Large numer active zones -> no overal failure
Large ‘quantal content’ per vesicle ~6,000 - 10,000 ACh mols
How is Pr similar to NMJ at CNS synapses?
- vast majority are L-glutamatergic or GABAergic
- 1 or few active zones per bouton (varicosity)
- small no. primed vesicles (2-10)
- miniature EPSPs ~0.1mV per vesicle
- quantal content smaller thsn NMJ (1,000-5,000)
Examples of improved reliability of release in CNS
1) Climbing fibre input from climbing olive -> Purkinje cells in cerebellum (1:1 relationship)
BUT mutliple active zones ~200, mutliple contacts/boutons, glutamatergic (EPSP~40mV), high Pr & many vesicles per active zone
2) sensory pathways: retinal ganglion -> dLGN (subcortical visual), anterventral cochlear nucleus -> trapezoid body (subcortical auditory)
- many AZs associated w/ each input axon fibre
Reliability can also be imporved by increase no. of AZs
e.g. CA1 pyrimidal cells have 3,000 excitatory & 1,500 inhib
What is peak Ca2+ produced by an AP dependent upon?
It affects Pr
Shape of AP - duration determines influx, targets for neuromodulation by activating GPCRs
Open probability & inactivation rate of VG Ca2+ channels, direct modulation by GPCRs (Gq vs Gi)
Increased Ca2+ conc during presynaptic receptor activation e.g. nAChRs, NMDA-Rs, Kainate-Rs
Increased Ca2+ conc during repeated Ca2+ channel activation - repeated APs & slow removal (residual Ca2+)
What is the conversion of Ca2+ signal to exocytosis dependent upon?
It affects Pr
No of primed vesicles at the active zone
Ca2+ responsiveness of these vesicles - Ca2+ sensitivoty of release machinery
What is synaptic plasticity?
Measured as: change in aplitude of postsynaptic response, synaptic strength or weight to same level of presynaptic activation
Increase or decrease
How can short term plasticity be demonstrated?
Paired pulse activation - amplitude of 2nd post synaptic response compared to 1st (increase or decrease)
Mechanisms of facilitation (input train)
Type of plasticity - elevated presynaptic intracellular Ca2+ conc
Residual presynaptic Ca2+ build up:
- influx of Ca2+ via VG channels, remains elevated for 10-100s
- slow buffering/reuptake (Ca2+ store in sER)
- influx during 2nd AP combines w/ 1st -> increases overall Ca2+ levels
- more likey to get vesciles to fuse over no. of synapses -> increases Pr
Ionotropic receptor activation:
- after 1st AP, NT release activates presynaptic receptors
- ionotropic receptors are Ca2+ permable cation channels -> elevate backgorund Ca2+
- influx during 2nd AP combines w/ original elevation -> increases overall Ca2+ levels
- increases Pr
e.g. kainate-Rs, NMDA-Rs, nAChRs
Mechanisms of depression
Type of plasticity (decreased)
Presynaptic vesicle depletion:
- each synapse has readily releasable pool (RRP), but slow replenishment after 1st AP
- period of reduced primed vesicles -> lower Pr
(typically when Pr high to start w/)
Presynaptic metabotropic autoreceptor activation:
- NT releases activate metabotropic autoreceptors
- GPCRs increase Ca2+ channel inactivation via By dimer of G protein
- reduces Ca2+ influx in 2nd AP
Postsynaptic ionoropic rceeptor desensitisation:
- some NT remains bound so receptors enter inactivated state
(typically when Pr high to start w/)
e.g. AMPA, nAChRs
Examples of short term plasticity within a train in the CNS
over ms-s time scale
Repeated activity by multiple equally spaced stimuli
Cerebellum - no lasting modification, climbing fibres depressed, parallel fibres facilitated, return to normal if impulses slowed
Hippocampus - CA1 response to Schaffer colateral activation -> initial facilitation then depression
What are the different types of memory?
Declarative (explicit) - facts, events
Non-declarative (implicit) - procedural, not conscious
Testing declarative memory (object recognition + placement, word recall increases CBF)
If hippocampus removed - declarative memory los, not procedural
Henry Molaison anterograe amnesia
Properties of hippocampal synapses under LTP
LTP - long term potentiation
Terje Lomo activated perforant input to dentate gyrus rabbit hippocampus.
Synaptic transmission monitored at low frequency stim <0.1Hz, then brief high 100Hz stim, then return to low.
-> Steeper rise time of EPSP + increased no. cells firing APs
Not a short term plastic change (enduring change)
In vitro LTP in hippocampal slices (CA3->CA1)
- recording form synpatic response at CA1 pyramidal neurones
- low frequency Schaffer collateral stim -> baseline EPSPs
- switch to 100Hz for 1 sec then back to low freq
-> increased synaptic strength/weight
4 trains at 100Hz results in robust LTP that is sustained vs ealry LTP from 1 train
What are the 3 component phases of long term potentiation?
Induction - initation LTP by 100Hz stim
Transient/early - reversible early-LTP following induction
Consolidated/late - permanent changes that then remain late-LTP
Role of NMDA-Rs during induction of LTP at CA3-CA1 synapses
Specific NMDA-R antagonist AP5 during HFS blocks LTP
NMDA-Rs blocked by Mg2+ when membrane hypeprolarised (jnternal negativity of membrane) SO only AMPA-Rs responsible for baseline transmission - does not lead to Ca2+ entry
- D-AP5 little effect of EPSPs evoked by <0.1Hz
Mg2+ block relieved by temporal summation -> depolarisation (high mem potential, -40mV)
- glutamatergic trnasmission voltage dependent so AMPA + MDA channels open
Role of NMDA-Rs in hippocampal spatial dependent memory
Morris water maze
Mice trained to swim to platform inn opaque liquid uisng spatial queues
- if treated w/ AP5 or KO, they cannot find the platform
- LTP sensitive to NMDA (contribute to declarative memory)
Protein kinases in early LTP
AP5 specific to induction to phase but protein kinase inhibitors (for PKC, CaMKII) prevents induction + early phase of LTP.
- LTP associated w/ increased phos of AMPA-Rs -> increases ion channel conductance (increased current)
- EPSP larger as it is prop to current
PKC + CaMKII target same phos site
PKC - protein kinase C
CaMKII - calmodulin dependent protein kinase II
Protein kinases in late LTP and the types of inhibitors used
PKA inhibitor (H89) transform perisitent long LTP -> shorter LTP, increases cAMP.
- PKA activation helps maintain high levels responsiveness
Anisomycin - translational inhibitor
Actinomycin D - transcriptional inhibitor, no new mRNA synthesised
-> suggest protein synthesis for long term LTP
nAChRs
Large family of pLGIC - cation channels permeable to Na+ & K+, some high perm to Ca2+
- fast excitatory transmission in motor neurones, skeletal muscle + autonomic ganglia, modulate release of dopamine in CNS
- 16 different subunits, foetal muscle has y instead of epsilon
Neuromuscular junction and structure of ACh/AChE
Motor neurone splits into finely branched synaptic nerve terminals/boutons -> endplate when contact skeletal muscle tissue
- muscle mem has junctional folds w/ nAChRs
ACh is ester of choline + acetic acid (ester bond & +ve charge)
AChE cleaves ester bond in synaptic cleft, collagen like tail (ColQ) that anchors AChE to basal lamina (3 ColQ + 12 catalytic subunits)
- in CNS has PRiMA (1 PRiMA + 4 catalytic subunits)
PRiMA - proline rich membrane anchor
Non-depolarising blockers
Competitive antagonists at nAChRs -> flaccid parlysis, overcome by AChE inhibitors
Atracuriam - immediate onset, non enzymatic spontaneous hydrolysis (30 mins)
Vecuronium/rocuronium - immediate onset (30-40mins)
Pancuronium - 2-3 min onset but much longer duration of action (100-200 mins), euthanasia + executions
administered IV
Depolarising blockers
Agonists at nAChRs -> fasciculation then flaccid paralysis, desesnitisation due to sustained depolarisation, mad worse by AChE inhibitors (neostigmine)
Suxamethonium - rapid onset, fast recovery (3 mins), hydrolysed by serum
- congenital abnormality (no or little serum ChE) -> prolonged block
Sugammadex
Drug that helps reverse block from non-depolarising blockers. Modified cyclodextrin (sugar) w/ lipophilic core that binds blocker + sequesters it.
- reversal deep block x17 faster than AChE
- quite expensive
Neuronal nicotinic receptors
Different structure to skel muscle but still pentameric.
e.g. 3xB2, 2xa4 -> may be important in nicotine addiction
e.g. 5x a7
ACh binding modulatory nAChRs activates Ca2+ influx -> depolarisation releases NT dopamine
Drugs targeting neuronal nAChRs
Varenicicline (champix) - selective partial agonist at a4B2 receptors (NICE says cheaper long term than nicotine replacement therapy)
Cytisine (from laburnam) used in E. europe w/ similar effects to varenicicline
Could ptoentially benefit parkinsons, AD, major depressive disorder, schizophrenia + pain (nicotine an analgesic)
- many clinical trials fail
BUT galantamine used for AD - allosteric modulator + AChEI, may als upregulate nAChR function
Botulinum toxins
ACt on SNARE proteins inhibiting vesicular release, from C. botulinum, LD50 = 2ng/kg
Targets cholinergic neurons in periphery - heavy chain recognises terminal + internalisation, light chain (proteolytic enzymes)
BoNT (A, C, E) - SNAP25
BoNT (C) - syntaxin
BoNT (B, D, F, G) - synaptobrevin
Causes botulism (food, wound, injection, inhalation), weakness, paralysis or resp muscles (death)
- no fever, still conscious
injected locally to overactive muscles
When are AChEIs useful?
When cholinergic deficit - block its degradation
1) reversal of NMJ block by non-depol blockers
- increases ACh so more to outcompete antagonists
- Myastehnia Gravis
- increased ACh compensates for loss due to internalisation, uses reversible (neostigmine, pyridostigmine) - AD
- overcome cholinergic deficit (donepezil, galantamine + rivastigmine used, all reversible)
- limited efficacy
Irreversible AChEIs
Organophosphates - developed as insecticides then as nerve agents (chemical weapons)
e.g. Sarin inhibits AChE at NMJs, ANS + brain -> repiratory failure
Attaches to serine residue on AChE forming strong bond so enezyme not recycled, Ser norally accepts acetate ro release free choline
-> desensitisation at NMJs, APs cease firing due to continuous depolarisation
Treatments to sarin
- injection atropine reverse effects at mAChRs
- pralidoxime recycles AChE (administered rapidly)
Sarin is slow ageing (5 hours ) vs Novichok A234 (short ageing, 2-4 mins) so resistant to pralidoxime
Novichok victims potentially saved w/ sedation by benzodiazepines (prevents CNS entry)
Myasthenia Gravis
Autoimmune attack on skeletal muscle - compromises motor endplate safety margin from NMJ overengineering.
95% patients have antiibodies targeting main immunogenic region (MIR, a.acids 67-76) on a-subunits (nAChRs)
1) receptors internalised when antibody binds
2) Destruction + simplification of enplate, complement attack reduces junctional folds, synapse widened
3) Antibody binding blocks ACh binding sites (competitive antagonism
Levels of MIR antibodies correlates w/ disease severity, 50% in humans
Diagnosis of Myasthenia Gravis
- Muscle weakness - drooping eyelids, slurred speech
- Testing serum for antibodies against nAChRs + MuSK
- Electromyography (EMG), activity of skeletal muscle, MG pateints show decline in size of AP after repeated stim
- Tensilon/edrophonium test, short acting AChEI given tosee if symtoms improved
MuSK - muscle specific kinase
Treatments of Myasthenia Gravis
mild MG:
AChEIs -pyridostigmine + neostigmine increases ACh in synapse counteracting loss of nAChRs (Mary Broadfoot Walker)
More severe MG:
Steroid drugs, azathriopine given to suppress immune response, increased risk of infection/cancer
Very severe MG:
Plasmaphereis / plasma exchange to remove autoantibodies from circulation.
Lambert Eaton Myasthenic syndrome (LEMS)
Leg muscles more heavily affected.
Targets presynaptic VG Ca2+ channels - fewer vesicles released after AP
- diagnosed w/ anti-Ca2+ antibodies
Treated w/ cancer tretament. Immunosuprression also used.
- pyridostigmine can boost cholinergc transmission
- 3,4-diaminopyridine increases Ca2+ in pre-synaptic terminals, more vesicles released
60% patients have small cell lung cancer
Congenital myasthenic syndromes (CMS)
Inherited disorders at NMJs, dominant or recessive.
- no autoimmune attack
- safety margin endplate compromised
- caused by mutant protein in synaptic transmission
-> vesicle packagin, docking, ACh synthesis, AChE deficiency, nAChR subunits (50%)
Autoimmune diseases not usually strongly heritable - some tendency genes associated w/ HLA variants.
Slow channel post synaptic CMS
Heterogenous dominant disorders - nAChR defects in skeletal muscle
-> simplified endplate + decreased nAChRs, widened synapse BUT no autoantibodies
Kinetic analaysis of V248F mutant in a-subunit (near leucine gate):
- receptor binds more tightly (lower k-1)
- once bound channel more like to open (increased B)
- channels close more slowly (decreased a)
a- G153S also changes closing behaviour + affinity for ACh (in N-terminal domain contirbutes to binding site)
- similar effect to V249F
-> produces mass cation influx into muscle, structral changes to endplate
Treatment - fluoxetine + quinidine, induce partial cation channel block
Mode switching
Low expression of a, B, delta & epsilon subunits incompatible w/ life.
If epsilon deficient - compensatory foetal y expression (less effective)
In HEk 239 cells:
Probability opening drops rapidly.
- insertion cytoplasmic loo of epsilon
- reduced epsilon expression
- compensatory expression of y
Fast channel CMS
epsilonP121L mutant - severe myasthenic syndromes BUT normal endplate structrue + normal AChR number
Openings more biref + far apart, kinetic analysis -> massive decrease in B so it binds ACh but cannot open.
Mutations to Pro alter gating + activation.
Recessive so need 2 copies
- epsilonG-8R signal peptide
- dletion of glycosylation site
Incomplete response to AChEIs
Other congenital LGIC chanelopathies
Startle syndrome - gly receptor mutations
Some epilepsies - GABA(A) + neural nicotinic receptors
Maybe some schizophrenias - mutants in neural nAChRs
Single channel kinetic analysis
Length of open time depends on k-1 (mean open time is 1/k-1)
Opening frequency depends on k+1 (mean closed time is 1/k+1)
Presence of multiple components -> multiple open states SO
mean closed time is 1/(k+1 + k+2)
In nAChRs:
Open, resting and desensitised states
- value of rate constants determines distributions of openings + closings
pattern governed by prob of k+1 = k-1
