Section A

Potassium Channels

Experimental Techniques

SCAM (substituted cysteine accessibility method) - Shows whether or not residues can be accessed by thiosulfonates which bind to the cysteine sulphide
Heterologous Expression (Xenopus oocytes)
Pharmacological blocking etc
Site directed mutagenesis

Channel Structure and General Information

Ion Channels

Role in Electrical Equivalent Circuit

These parallel resistors and their placement are the main determinants of neuron excitability and firing pattern

The presence of VG channels in the membrane allows transverse flow of charged ions in response to changes in membrane potential
Cells are able to generate rapid signals which can propagate and encode much greater information than equivalent molecular forms of communication (paracrine endocrine etc) would allow

Type-A K channels (voltage gated, like Shaker) are responsible for the generation of the action potential while others are responsible for setting and stabilising the resting membrane potential (such as IRK and Tandem-Pore channels)

Sequencing the first channels

Sodium Channels (and Ca2+)

Potassium Channels


Some potassium channels show inwardly rectifying currents

Attributable to polyamine block (e.g. spermine) and Mg block which both show strong voltage dependence
Minimal currents generated at depolarised potential rather than linear IV relationship
Structure also contributes to preference for inward flow of K+ ions, to reverse hyperpolarisation and restore the resting membrane potential


Calcium-Activated such as BK (CBTx sensitive)
A-type = voltage-gated transient outward-rectifier K+ channels

Mackinnon and crystallography of the K channel
4 subunits of a tetramer
in KcsA, 2 TM s.u.s with inner helix, outer helix, pore helix and selectivity filter
12 - narrow selectivity

Glutamic acids at channel mouth
then into wide hydration chamber
then into selectivity filter
then experiences negatively charged aspartic acids and enters the extracellular space

Permeation and Toxins


Charybdotoxin, TEA, Kappa-Conotoxin?

Scorpion Toxins

Induced fit?

2002 Erikson

Lange 2006


Tetraethylammonium (TEA)

Binds to internal side of selectivity filter within the aqueous chamber; blocks the dehydration transition site indicated by analysis indicated with caesium (has lowed dehydration energy and so has better stability at the transition site).

Internal sensitivity is voltage dependent due to block of intracellular side by ball-and-chain preventing TEA acccess

And Inactivation; Foot in the door

(and difference between Kcsa and Shaker??)

Importance of extracellular block mechanism; demonstrating C-type inactivation (studied often in Shaker or Kcsa chimeric channels with removed N-terminals to negate N-type inactivation)
Depends on same residues as TEA binding (477 Tyrosine or Phenylalanine aromatic residues
Thought to prevent “pinching” of pore through interaction with hydrophobic residues
TEA inhibits slow inactvation in Wt shaker channels





Ahern et al 2006 (questioning Kcsa orientation being applied to Shaker)

Tested for cation-pi in Shaker channels using in vivo nonsense suppression; increased fluoro of Phe side-chains at position 449
Minimal steric disturbance
Varying degrees of fluorination of aromatic side-chain to withdraw pi electrons and weaken cation-pi progressively, without altering hydrophobicity
Also progressively increased K(i) for TEA block of Shaker

In follow up 2009

Demonstrated that N-type inactivation of Shaker channels involves collapse of the outer edge of the external vestibule


MacKinnon and Miller 1989 - Shaker (A-type)

Further Mutagenesis Identifies the P Loop

Yellon et al (MacKinnon), 1991

Chimeric Mongrels

Hartmann et al 1991


K Channel Selectivity

Zhou et al, (MacKinnon) 2001

Data from Review

Comparison to Ca channels


N-Type Inactivation

Mutagenesis of the Ball and Chain

Round 1

Round 2 - Lets get selective

What was the purpose of the processding residues?


Zagotta, Hoshi and Aldrich et al 1990 Paper 2


TEA and Slow inactivation

Choi et al 1991



The Paddle Model

FRET and Rotation



Lateral Knowledge

The HCN Channel and others relevant to Qs

Hyperpolarization-activated cyclic nucleotide-gated channels are intermembrane proteins that serve as nonselective LGIC
Help to generate rhythmic activity in heart and brain cells
Responsible for Ih or If (for funny) current

History and Method

The Voltage Clamp

The Process

The Assumptions

Charged gate assumption was made to confer the voltage sensitive changes in ion conductance observed.


The Model

Gating and Energy

The basis

Extolled in the early papers

The Formulae

Number open = n= α/(α + β)

At steady state (Vm is constant) dn/dt (change in number of open channels) is constant. Though there may be initial variation in ion flux this will eventually balance as ion channels inactivate etc.

α and β (rate constants of opening) are voltage sensitive.
In response to a voltage step,

n_t=1−e^((−t)/τ) τ=1/(α+β)

Sodium channel kinetics on the other hand are less straight forward due to rapid inactivation. Modelled using m3h (inactivating h gate).


The discussed m/h/n are dynamic variables which denotes the probability of “opening”; m and n are positively related to membrane potential while h decreases with depolarisation. h and n are both slow in their kinetics however. These factors are essential to the action potential.

These factors with their voltage dependence are used to express conductance in voltage dependent terms

The final equation is obtained by combining conductance questions with the equation for current (factoring in driving force)
Crucial to include static leak current
Gives an equation for change in membrane potential over time in response to external current


Explains the absolute refractory period observed;

Also afterhyperpolarisation supports trains by reducing the threshold;


Sodium Channel Inaccuracies

Independence of gates

Sodium channel inactivation


Method to Madness

NT Release

Fatz/Katz/del Castillo

Katz showed

Katz’s questions
1) How does the synaptic vesicle fuse with the plasma membrane
2) How is this controlled by Ca2+
3) How is the rapid coupling of an AP and Ca2+ to NT release achieved?

Membrane Fusion

Theory of fusion; Stalk hypothesis


Knowledge of fusion proteins found through viral fusion protein led to Jackknife model

Fusion Apparatus; SNAREs

Developing the SNARE Hypothesis

Energy requirement for unfavourable membrane fusion immediately indicated the involvement of an ATPase
Rothmandeveloped cell free fusion assays of vesicles, and used poisons which blocked fusions
Used them to identify critical proteins
Identified NSF (NEM-sensitive fraction) which has ATPase activity, as well as SNAPs
Previously found by Schekman using radiation of yeast that mutation of the gene Sec18 blocked release. Turns out NSF and Sec18 are the same
Used bead purification column with brain homogenate and NSF with unhydrolysable ATP-gamma- to freeze the reaction
Bound proteins constituted key parts of release machinery
Identified SNAREs (R-SNARE Synaptobrevin/VAMP and the (usually) Q(t)-SNAREs syntaxin and SNAP-25)

Interestingly Botox (clostridial toxin) is a zinc protease which targets and cleaves SNAREs, disabling NT release.

SNARE hypothesis supported by using N-terminal labelling and Electron microscopy to show that N terminals come together. DUe to the fact taht SNARE C-terminals are always in the membrane though SNAP-25 has no TM domain; it is attached to the membrane by palmitoyl side-chains which are covalently bound to cysteines.



Alternative Fusion models

Accessory Proteins

SM proteins (Munc18)


Cong Ma et al., 2011

Rabs and Rims

RIMBP and RIM bind to Rab to bring vesicle to membrane
Complex also binds Munc13 which binds to syntaxin-Munc18 to open the Qa-SNARE
SNARE-pin assembles
Is stabilised by Munc18
Reliance on various proteins maintains focus in active zone, at calcium channels


Sudhof 2013


Proteins producing Ca-dependence and synchrony

Synaptotagmin - calcium sensor


Ca-dependent Synaptotagmin Isoforms - 2/7/9


Additional role

The Complexin Clamp

Questions and Answers

Section B

Quantal Theory of Release


Synaptic transmission relies on three main elements

  1. Presynaptic calcium
    • Can be studied using calcium chelators and indicators
  2. Vesicle pool
    • Capacitance measurements (addition of vesicle to membrane) and imaging vesicle proteins with pH-sensitive dyes
  3. Postsynaptic receptors
    • Pharmacological manipulation using antagonists and allosteric modulators can show changes in postsynaptic receptor saturation


Quantifying the elements


R - postsynaptic response
N - maximum number of readily releasable vesicles/release sites
Q - average quantal response (normally univesicular)
P - mean probability of release

The product of N and po describes Nt (total number of readily releasable vesicles at t)


Best model so far (though has been improved by modifications since). However the principle failing is in that rarely is this able to include post-synaptic influences.

Additionally, quantal parameters are complicated by issues such as receptor saturation

The Role of Calcium Domains

There can also exist heterogeneity within a single synapse

Cluster Perimeter Release Model

Nakamura et al 2015

VgCC clustering at active zones shown using immungold labelling of freeze fracture preparations

Cluster area (not density) increases with age

Accounts for

Using simulation with structure of cluster indicated by above information, found that EGTA inhibition could only be reproduced in calcium sensory was at the edge of the cluster

Change in EGTA sensitivity due to increase in cluster along with reduced coupling distance leading to greater spike in [Ca] along with location closer to higher gradient.

So transition from microdomain to nanodomains due to increased clustering of VgCCs and tighter linking of vesicles to the perimeter of these clusters

Post-Synaptic Elements

Multi-Vesicular Release

Additionally, glial ensheathment can isolate synapses, with high numbers of AA transporters to terminate glutamate signals rapidly

Also some AMPA-R isoforms recover rapidly from desensitisation

Quantal Release and STP


Residual calcium hypothesis

Use-dependent replenishment



For example, STD allows neurons to respond to relative changes

Depression allows detection of changes to low frequency inputs.

Therefore Synaptic STP allows performance of powerful computations through (at least) 2 main mechanisms

Computational importance of STPlas

Within a functional context

Gain Modulation

Gain modulation is the change in slope in I/O relationship
Important in terms of functional connectivity of neurons
Controls routing of information along a network
Model pyramidal neuron without STD shows that inhibition produces lateral movement of I/O firing rate relationship
Addition of STD enables gain modulation leading to more complex changes and inhibition producing a reduction in maximum firing rate
Neuronal activity is more information rich.

*Based primarily on theoretical considerations, facilitation is thought to influence both information transfer and network dynamics profoundly.

Measuring STPlas

Different methods

Paired-Pulse Ratio

Normally deliver second pulse at different intervals to assess plasticity time course

<1 the has undergone PPD, >1 then PPF

Post-Tetanic Ratio

More stimuli are able to induce additional, longer lasting forms of plasticity
Similarly, varying intervals of test pulse allows description of plasticity time course.

Steady State Ratio

Forms of Potentiation


Mechanisms of STPlas

Always multiple in a synapse
High release probability leading to depletion of the RRP will almost always lead to depression,



Frequency Stimulation and Presynaptic Calcium channel

Katz et al first to propose that build-up of Ca in active zone boosts release

A molecular mechanism

Calcium and Cadmium

Olesckevich and Walksey 2000

Fernandez-Chacon et al 2001


Presynaptic PPD largely though to be due to a reducion in RRP

Residual calcium can acccelate recovery from PPDepression

Addressing assumptions


Will all modulate Q

Receptor Saturation

Masks Pre-synaptic Multi-vesicular release and alters PPR at climbing fibre synapse

Receptor Desensitization

Interplay between P and Q


Polyamine block

Desensitized AMPA receptors can diffuse away from the cleft?
However data indicates that contribution would be very small

LT Plasticity and STDP??


Donald Hebb and the Hebbian Postulate

When an axon of cell A is near enough to excite a cell B and repeatedly or persistently
takes part in firing it, some growth process or metabolic change takes place in one or both
cells such that A’s efficiency, as one of the cells firing B, is increased.
Cells that fire together wire together

STDP factors in element of “Reliable contribution”


Physiological evidence to support the Hebbian postulate has accrued over the years

Bliss and Lomo 1973 - LTP

Dunwiddie and Lynch 1978 - LTD

Kirkwood et al 1993 - Input Pattern and cooperativity


Bliss and Collingridge 1993 - Support for the Hebbian Postulate

Three features of LTP

  1. Cooperativity
    • Induction by coincidenct activation of synapses conveying subthreshold inputs
  2. Associativity
    • Can potentiate weak input which it coincides with a strong input
  3. Input Specificity
    • Can only be induced at activating synapses
    • Due to compartmentalized (microdomain) increase in Ca2+ through unblock of NMDA-Rs

Importance of Coordination - STDP

Markram et al 1997 - Synaptic Efficacy controlled by coordination of EPSPs and APs

Functional Implications of STDP

From Hebb to STDP

With hindsight, it is clear that a temporal element to the Hebbian Postulate is required
If two neurons fire simultaneously then the presynaptic neuron can not have contributed to the firing of the postsynaptic
Therefore a strengthening of the connection would have no computational value
This bidirectional element of STDP combines perfectly with the heart of the Hebbian Postulate
Of strengthening Cause and Effect; enhancing signal and reducing the noise.

Alternative simplification would maybe be
“Those who trigger each others firing, strengthen their wiring”

Perhaps Slightly more wordy, yet includes the causative elements of “firing together” that STDP demonstrates

Cellular and Molecular

All well and good stating that it occurs; but what are the mechanisms?

Evidence for the role of bAP

The bAP is relatively recently acknowledged phenomenon
Generation of action potential in the soma generates an “echo” which depolarizes parts of the dendritic tree
Activates VGNa and Ca Channels
Depolarizes the dendritic tree

Stuart and Sakmann 1994

bAP and the EPSP

Implications for LTP are complex
Whole cell mechanism of potentiating inputs by reducing EPSC required to reach threshold potential
Additionally occurrence of calcium spikes is likely to activate post-synaptic mechanisms, potentially affecting receptor density etc.

Coincidence Detection in tLTP

Rodriguez-Moreno and Paulsen 2008 - Mechanisms of coincidence detection

Presynaptic Effects?

Mechanism of Presynaptic LTP: REVIEW (R-M et al 2010)

Signal Transduction

Post-synaptic calcium seems key; conventional STP model involves Ca gradient model
LFS leads to low rise in [Ca]
HFS produces large increase
Produce LTP and LTD respectively
Mg block removal by bAP likely contributes to a similar mechanism in STDP


CaMKII (Calcium/Calmodulin-dependent Kinase II)

Recylcing of AMPARs



Computational implication from a Model

Section C


RL focus on tonic GABA-A

Aetiology of Epilepsy


Network Changes underlying propensity to seizure


Network Level

Modify Gain leading to increased responsiveness?
Steeper slop in I/O relationship

Loss of Interneurons Changes Inhibitory Pattern

Kobayashi and Buckmaster 2003

Increased Tonic GABAA

Role of Tonic inhibition in epilepsy indicated by mutations in genes encoding extrasynaptic GABA-ARs associated with epilepsy
Loss of phasic dendritic inhibition as explained above

Pavlov and Walker Review/Scimemi 2006


Changes in tonic inhibition along with decrease in phasic inhibition leads to a network with reduced excitability as well as a narrower dynamic range, combined with decreased stability (tonic inhibition does not affect neuronal gain, mainly subthreshold effects).

However increased tonic GABA-A activation could be pro-convulsive due to excessive load on Cl- extrusion combined with decreased KCC2 expression
Pushes reversal potential to more depolarised values


Cell Intrinsic

Bursters *

Sanabria 2001

Enhanced spontaneous activity

Bernard 2004

Shah 2004

Reversing excitability with Kv1.1 and Seizures with halorhodopsin

Wykes 2012 - The epileptic neuron

Cell Synaptic

Increased Glu release from astrocytes

Tian 2005

AMPA/Kainate Changes

Epsztein 2005

Enhanced NMDAR

Lieberman and Mody 1999 - Enhanced NMDAR

Scimemi 2006 - Enhanced release and NMDA

Depolarising GABA-A; Return to Immaturity


Mechanosensation, Pain and Sodium Channels

Pain Transmission and Peripheral Nerves

Pain transmission; A delta fibres

Blocking Pain Transmission blocks Pain

Haroutounian et al 2014

Varo et al 2014

Modality Specificity of Primary Afferents

Karolinska institute bank account is hench

Supported by Usoskin finding distinct classes of sensory neurons
Unbiased classification to produce mocelular map
11 unique types of sensory neurons
Vary in terms of receptors, channels, trafficking proteins and peptides
all indicate different roles

Conduction of Sensation

Sodium Channel Morphology

Structure of Na Channels


Alpha Subtypes

Alpha subtypes

Sodium Channel Alpha Subtype Distribution

Abrahamsen et al 2008 - Mouse

Role in Sensitization

Laird and Cervero 2002

Also shown separately that; increased trfficking of Nav1.8 caused by PGE2 contributes to inflammatory sensitization



Fertleman 2008 - PEPD (familial rectal pain)

Primary Erythromelalgia

First channelopathy linked to chronic neuropathic pain (2004) Similarly to PEPD, flushing and episodes of intense pain
Burning pain specifically (TRPV1 expressing neurons?

KO showing function

Global Nav1.7 deletion leads to early death in mice

Nassar et al 2004

Minett et al, 2012

Failure in Cancer Pain

Minett and Falk, 2013

SCN9A LoF leads to peripheral enkephalin production along with attenuation of electrical excitability
Together lead to loss of pain


Nociceptors and the Action Potential

Lucas 1909 showed all or nothing signalling using current input and muscle movement
Cole and Curtis 1939 showed Action Potentials linked to increases in conductance
Hodgkin and Huxley used voltage clamp to detect changes in current, attributed to Na and K ions mainly linking potential change to equilibrium potentials for major ions Na, Ca, K and Cl. Developed gate theory of channel activation and inactivation which determine the kinetics of the action potential.

Voltage Clamp, allows study of ionic changes in media
Excitable tissue

At rest, K+ permeable, Na impermeable though large driving force
Raising extracellular K+ depolarises membrane potential. Increasing excitability of the neuron

Axon Hillock; site of AP initiation (

  1. Lower threshold for AP initiation due to high density of Nav (20-200 x more than Soma/dendrites)
  2. small diameter , reducing current required to drive membrane threshold
  3. Different Nav channel properties

Propagates, as distal Na channels activate, proximal Na channels inactivate and late K channels activate
This active current generation is essential for the all or nothing current generation behind the action potential

Pharma tools in ion channels

History of Na channel comprehension

Treating Pain

Molecular Aspect of Sensing



Can be measured experimentally through different methods of inducing stretch of neuron membrane

Mechano-Transducers; Piezo channels


(Coste et al 2010)

Patapoutian Group showed that Piezo2 is crucial in innocuous touch but not in noxious mechanical sensation though inverse in drosophila;

Side-note; TRPV1 activation inhibits Piezo receptors through depletion of PIP and PIP2

Surprise; TRPA1

More Surprises TRPM8

Molecular Sensitization; Allodynia and Hyperalgesia

Galeotti et al, 2004 - Protein Kinase C

Di Castro et al., 2006 - NGF and PKC

Anything with a banging essay