Membrane excitability Flashcards

Question

Impact of capacitance on membrane

Answer 1

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.

Answer 2

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 ## Footnote both effects interact

Answer 3

1st H+H described Na+ & K+ in giant squid axon 1950s -> coordiantes water ejection from mantle cavity in escape response ## Footnote H+H - Hodgkin & Huxley

Answer 4

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.

Answer 5

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+) ## Footnote In one experiment H+H used ion substitution - choline+ used instead of Na+ -> early inward current disappeared

Answer 6

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

Answer 7

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 ## Footnote y is single channel conductance Vm - Erev is driving force

Answer 8

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 ## Footnote i = sum of microscopic currents

Answer 9

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

Answer 10

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 ## Footnote lipid permeability increased at higher pH -> e.g. cocaine chewed w/ alkaline

Answer 11

Saturable process - according to M-M kinetics Has Jmax & Km

Answer 12

Transport 1 or more substrate (1 against a gradient) - enzymatic activity (ATPase) for primary AT e.g. Na+/K+ pump 3Na+ out 2K+ in

Answer 13

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

Answer 14

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

Answer 15

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)

Answer 16

NHE for Na+/H+ exchange (pH) AE (anion exchanger) for Cl-/HCO3- exchange

Answer 17

Amphetamines -> NET (SLC6A2) & DAT (SLC6A3 for dopamine) SSRIs - inhibitors of Na+/serotonin co-transporter (SLC6A4) New: SGLT2 inhibitors for diabetes ## Footnote SSRI - Selective serotonin reuptake inhibitors

Answer 18

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

Answer 19

Maintain cell volume - pre dates + gives rise to membrane potential Transport influenced by both chemical gradient & membrane potential.

Answer 20

Convert chemical gradient to an electrical gradient EC gradient = Vm - Ex Ex is Eq for ion x.

Answer 21

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

Answer 22

Net gain on K+ & Cl- using NKCC1, very slow. Driven by Na+ gradient, Na+ removed by ATPase. Water movement in via osmosis.

Answer 23

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-

Answer 24

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

Answer 25

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

Answer 26

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 ## Footnote pH = -log10 [H+], optimum [H+] = 20-100nM

Answer 27

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 ## Footnote ASIC - acid sensing ion channel

Answer 28

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 ## Footnote e.g. in cardiac myocytes, dye attached to lipid permeable ester so it can diffuse into cell - then hydrolysed so trapped in cytoplasm

Answer 29

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

Answer 30

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 ## Footnote NHE - Na+/H+ exchanger NBC - Na+/HCO3 co-transporter CHE - Cl/OH exchanger CBE - Cl/HCO3 exchanger

Answer 31

- 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

Answer 32

electroneutral - non et movement of charge e.g. NHE, NKCC1 electrogenic - net movement of charge generating small electrical current e.g. SERCA, SGLT1/SLC5A1

Answer 33

Cell: VGCCs, NMDA-Rs, store operated Organelles: IP3 GPCRs, ryanodine Extrusion + sequestration effective off switches

Answer 34

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

Answer 35

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 ## Footnote other CNS barriers inc retinal + blood-spinal chord

Answer 36

Increased IP3 via Gq increases [Ca2+] Loss of K & Cl decreases cell volume HCO3- efflux increases [H+]

Answer 37

Linked by junctional complexes (claudins + occludins) Claudins determine tightness + selectivity of junctions (paracellular transport) Transcellular transprot requires polarity.

Answer 38

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) ## Footnote Cerebrospinal fluid (CSF)- transport of substances to the brain + drainage of waste products/toxins

Answer 39

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.

Answer 40

Chronic - MS, autoimmune encephalomyelitis, AD Acute - ischaemic stroke, hypertension, seizure -> associated w/ misregulation of tight junctions or pumps/carriers

Answer 41

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.

Answer 42

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)

Answer 43

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 ## Footnote Reese + Karnovky contend barrier function at endothelia level not astrocytes

Answer 44

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

Answer 45

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

Answer 46

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 ## Footnote genetic diversity underlies ion channel diversity - subtypes expressed according to cells requirements

Answer 47

- calcium activated - inwardly rectifying - two pore domain - voltage gated

Answer 48

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 ## Footnote 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

Answer 49

All related. S5-P-S6 motif in pore lining. - some have charged a. acids -> voltage sensors

Answer 50

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

Answer 51

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+

Answer 52

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

Answer 53

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

Answer 54

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 ## Footnote encoded by same gene family

Answer 55

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

Answer 56

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)

Answer 57

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.

Answer 58

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. ## Footnote L type are dihydropyridine sensitve vs other are not -> early classification

Answer 59

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)

Answer 60

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

Answer 61

Ziconotide (synthetic) blcoks channel + reduces NT release for chronic pain.

Answer 62

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).

Answer 63

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) ## Footnote Hetermoeric channels activated by smaller depolarisations (more powerful)

Answer 64

Control of AP duration - short if current activates fast (nerves/skl muscle) - long if current actuvates slow (heart) ## Footnote Pharma - blocked by quaternary ammonium ions (TEA)

Answer 65

Controls AP interspike interval - short if current small - long if current large ## Footnote Pharma - blocked by 4-AP (aminopyridine) -> dendrotoxin

Answer 66

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. ## Footnote Pharma - blocked by charybdotoxin, iberiotoxin + TEA

Answer 67

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. ## Footnote Pharma - SK blcoked by apamin, IK blcoked by charybdotoxin + TEA

Answer 68

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

Answer 69

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

Answer 70

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

Answer 71

Heart: Sa node - pacemaker activity in HCN Cardiac myocytes - ventricular AP Pancreas: Insulin secreting B-cells - sensing glucose (Kir 6.2)

Answer 72

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 ## Footnote Same in Av node

Answer 73

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

Answer 74

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)

Answer 75

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.

Answer 76

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)

Answer 77

HCN blocker slows HR -> for heart failure w/ elevated HR, chronic stable angina (insufficient myocardial perfusion)

Answer 78

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

Answer 79

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

Answer 80

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 ## Footnote hERG - human Ether-a-go-go related gene Kv 11./ Kr

Answer 81

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

Answer 82

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 ## Footnote ATP-sensitive K+ channel acts as 'glucose sensor' - found in other excitable cells

Answer 83

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 ## Footnote forms K atp

Answer 84

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 ## Footnote LoF allows Ca2+ influx but GoF does not

Answer 85

- 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)

Answer 86

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

Answer 87

Small mols: Amino acids - gly, GABA, glu Amines - NA, dopamine, serotonin, ACh Purines - ATP, adenosine Peptides- endorphins, neuropeptide Y, somatostatin, substance P

Answer 88

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)

Answer 89

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

Answer 90

- 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) ## Footnote Vesicles derived from ER + trafficked to docking sites.

Answer 91

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)

Answer 92

Full fusion not required - NT leaks out of small fusion pores - SSVs recycled intact from cell mem + not via clathrin coating via endosome

Answer 93

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)

Answer 94

1. Docking- vesicle docks at active zone (differ by vesicle no. between neurones) 2. Priming - vesicle maturation + made competent, requires ATP, protein conf changes drives release 3. Fusion/exocytosis - full fusion of mems requires Ca2+ & involves Ca2+ sensor protein, NT discharged ~1msec 4. Endocytosis - recovery of fused membrane, recycling triggered by intracellualr Ca2+, skeletal lattice formation (clathrin monomers), ~5 secs + ATP dependent 5. Recycling - conserves vesicle mem via endosome, refill w/ NT -> ATP-dependent ## Footnote DPFER

Answer 95

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

Answer 96

Synaptobrevin, syntaxin, SNAP-25 make 1:1:1 trimeric complex -> coiled coil quaternary structure of a-helices from SNAP-25

Answer 97

Snares form tighter complex -> 'zippering' forms SNARE-pins - ATP-dependent - assisted by Munc18s, binds to syntaxin Habc domains

Answer 98

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

Answer 99

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 ## Footnote Pr is low, fusion failure rate is high

Answer 100

Large numer active zones -> no overal failure Large 'quantal content' per vesicle ~6,000 - 10,000 ACh mols

Answer 101

- 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)

Answer 102

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

Answer 103

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+)

Answer 104

No of primed vesicles at the active zone Ca2+ responsiveness of these vesicles - Ca2+ sensitivoty of release machinery

Answer 105

Measured as: change in aplitude of postsynaptic response, synaptic strength or weight to same level of presynaptic activation Increase or decrease

Answer 106

Paired pulse activation - amplitude of 2nd post synaptic response compared to 1st (increase or decrease)

Answer 107

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

Answer 108

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

Answer 109

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

Answer 110

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 ## Footnote Henry Molaison anterograe amnesia

Answer 111

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)

Answer 112

- 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

Answer 113

Induction - initation LTP by 100Hz stim Transient/early - reversible early-LTP following induction Consolidated/late - permanent changes that then remain late-LTP

Answer 114

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

Answer 115

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)

Answer 116

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

Answer 117

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

Answer 118

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

Answer 119

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) ## Footnote PRiMA - proline rich membrane anchor

Answer 120

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 ## Footnote administered IV

Answer 121

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

Answer 122

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

Answer 123

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

Answer 124

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

Answer 125

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 ## Footnote injected locally to overactive muscles

Answer 126

When cholinergic deficit - block its degradation 1) reversal of NMJ block by non-depol blockers - increases ACh so more to outcompete antagonists 2. Myastehnia Gravis - increased ACh compensates for loss due to internalisation, uses reversible (neostigmine, pyridostigmine) 3. AD - overcome cholinergic deficit (donepezil, galantamine + rivastigmine used, all reversible) - limited efficacy

Answer 127

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

Answer 128

- 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 ## Footnote Novichok victims potentially saved w/ sedation by benzodiazepines (prevents CNS entry)

Answer 129

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 ## Footnote Levels of MIR antibodies correlates w/ disease severity, 50% in humans

Answer 130

- 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 ## Footnote MuSK - muscle specific kinase

Answer 131

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.

Answer 132

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 ## Footnote 60% patients have small cell lung cancer

Answer 133

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%) ## Footnote Autoimmune diseases not usually strongly heritable - some tendency genes associated w/ HLA variants.

Answer 134

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 ## Footnote Treatment - fluoxetine + quinidine, induce partial cation channel block

Answer 135

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

Answer 136

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

Answer 137

Startle syndrome - gly receptor mutations Some epilepsies - GABA(A) + neural nicotinic receptors Maybe some schizophrenias - mutants in neural nAChRs

Answer 138

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 ## Footnote pattern governed by prob of k+1 = k-1