Action Potentials

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Action Potentials

• Currents underlying the Action Potential
• Voltage clamp
• Action potential propagation
• Other currents
• Kinetic properties
• Variation in firing patterns

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What is Action Potential?

• Depolarization to Sodium Reversal Potential
• pK pNa 0.05
• Repolarization
• Hyperpolarization
• pK pNa 20
• Repolarization to resting potential

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Squid Giant Axon
0.5 mm Diameter, with 0.1 mm pipette
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Action Potential Currents

• Change in membrane potential accompanied by
• Increase in conductance
• Early increase in sodium conductance causes
  depolarization
• Later increase in potassium conductance causes
  after hyperpolarization (AHP)
• How are currents evaluated?

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Voltage Clamp

• Measure VM Inside - Outside
• Choose Clamp potential (VC)
• Calculate VC - VM
• Inject current k (VC - VM)

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Voltage Clamp

• If VC gt VM, injected current is positive
• Membrane potential increases
• VC - VM decreases
• If VC lt VM, injected current is negative
• Membrane potential decreases
• VC - VM decreases

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Voltage Clamp Currents

• Early inward current
• Negative current injected by clamp to maintain
  membrane potential.
• Negative current is compensating inward flow of
  positive ions
• Transient current
• Later outward current
• Positive current injected to compensate for
  outward flow of positive ions
• Persistent current

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• Fast, transient current carried by sodium ions
• Delayed, persistent current carried by potassium
  ions
• Very fast (lt 1 ms) capacitive current (not shown)

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Action Potential Currents

• Observed after blocking 1 current with toxin
• Potassium current
• increases with depolarization
• Sodium current
• increases with small depolarizations,
• then decreases with larger depolarizations,
• then reverses (changes from inward to outward)

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Action Potential Conductance

• Potassium channel
• Conductance increases with depolarization
• Driving potential increases with depolarization
• Sodium channel
• Conductance increases with depolarization
• Driving potential decreases with depolarization

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Action Potential Conductance
Reaches maximum value with increasing
depolarization
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Terminology

• Activation
• Turning on of current with depolarization
• De-activation
• Turning off of current with repolarization
• Inactivation
• Turning off of current with sustained
  depolarization
• De-inactivation
• Removal of inactivation (block) by repolarization

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Action Potential Channels

• Voltage dependence mediated by gates
• Hypothetical construct by Hodgkin and Huxley more
  than 50 years ago
• Existence of gates, physical structure of gates
  observed only recently
• Activation gates open with depolarization
• Inactivation gates close with depolarization

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Potassium Channels

• Squid Potassium channel has one type of gate
• Gate opens with depolarization
• Gate closes with hyperpolarization
• Current continues to flow with sustained
  depolarization

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Sodium and Potassium Conductance
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Sodium Channels

• Sodium channel has two types of gates
• Activation gate
• Opens with depolarization
• Closes with hyperpolarization
• Inactivation gate
• Closes with depolarization
• Opens with hyperpolarization
• Current flows when both gates open

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Sodium Channels

• Depolarization causes activation gate to open
  quickly
• Current flows
• Depolarization causes inactivation gate to close
  slowly
• Current stops flowing
• Repolarization required to de-inactivate channel,
  allow subsequent current flow

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Sodium Current
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Action Potential Generation

• Depolarization of membrane initiates process
• Sodium channels open
• INa causes depolarization
• Positive feedback causes rapid depolarization
  toward ENa
• Depolarization must be sufficient
• INa greater than "leak" currents

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Action Potential Repolarization

• Depolarization causes
• Sodium channel inactivation
• Potassium channel activation
• When IK exceeds INa, membrane repolarizes
• VM hyperpolarizes
• De-activation of IK is slow process
• When VM reaches VR, IK still active

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Action Potential Threshold

• Transforms analog value of membrane potential
  into digital all-or-none action potential

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Refractory Period

• Time after action potential when another action
  potential can't be produced

• Absolute refractory period
• INa is still inactivated
• Relative refractory period
• AP is possible with higher current injection
• Due to persistence of IK

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Action Potential Propagation

• Inward current flows forward and backward in loop
• Forward current depolarizes membrane
• Depolarization initiates action potential
• Backward current flow encounters refractory part
  of membrane

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Action Potential Propagation
Backward current encounters refractory membrane
Forward current Depolarizes membrane
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Firing Patterns

• Neurons exhibit distinct electrophysiological
  properties
• Pattern of repetitive action potentials called
  firing pattern
• Regular Firing
• Adaptation
• Bursting
• Delayed Firing

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Regular Firing

• Repetitive action potentials in response to
  sustained current injection
• Increase in firing frequency with increase in
  current amplitude
• Firing continues with sustained current

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Firing Patterns

• Spike Frequency Adaptation
• Synonymous with accommodation
• Decrease in frequency with time
• Most pyramidal neurons, cortical and hippocampal
• Delay
• Time delay between depolarization and firing
• Time delay called latency
• Seen in spiny projection neurons

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Firing Patterns

• Bursting
• Clusters of action potentials
• Some pyramidal cells and thalamic relay cells
• Spontaneous firing
• Also called beating
• Action potentials generated regularly with no
  inputs
• Seen in neuromodulator releasing neurons

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Channel Function

• Drugs which block specific channels
• Compare activity with and without drug
• DNA manipulations
• Delete or replace normal gene with mutated gene
  to prevent normal channel function
• Insert dominant negative gene into genome to
  prevent expression of normal channel
• Link gene expression with tetracycline, to
  produce inducible mutation

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Sodium Currents

• 10 different genes encoding a subunits
• Nav1.4, 1.5 found in muscle
• Nav1.7,1.8,1.9 found in peripheral nervous system
• Nav1.1, 1.2, 1.4, 1.6 found in central nervous
  system
• Nav1.2, 1.6 are in Nodes of Ranvier, axon initial
  segment

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Sodium Currents

• Transient, INaF
• Responsible for Action Potential
• Blocked by TTX
• Persistent, INaP
• Kinetics
• Does not inactivate
• Threshold near resting potential
• Controversy over whether INaP is a window current

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Toxin Binding Sites

• Picture from sigma catalog of blockers

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Window Current
Activation shifted to higher potential, no window
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Sodium Currents

• Function of Persistent
• Enhancement of subthreshold synaptic potentials
• Depolarization activates INaP, gt more
  depolarization
• Hyperpolarization de-activates INaP, which
  produces more hyperpolarization
• Plateau potential
• Prolonged potential that remains after synaptic
  inputs or current injection is removed
• Contributes to persistent firing

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Calcium Channels

• Channels permeable to calcium
• Inward current in the absence of sodium
• Can generate action potential
• Slower and wider than sodium spike
• Adds to depolarization of spike
• Allows calcium entry during spike
• Involved in burst firing
• Multiple types of channels
• Importance due to role of calcium ions as second
  messengers

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Calcium Channels - 10 a subtypes

• Gene
• Cav1.1
• Cav1.2
• Cav1.3
• Cav1.4
• Cav2.1
• Cav2.2
• Cav2.3
• Cav3.1
• Cav3.2
• Cav3.3

• Channel subtype
• L High voltage activated
• L High voltage activated
• L Low voltage activated
• L High voltage activated
• P/Q High voltage activated
• N High voltage activated
• R High voltage activated
• T Low voltage activated
• T Low voltage activated
• T Low voltage activated

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Calcium channels

• L type channel
• Long lasting (little to no inactivation)
• High threshold -20 to -10 mV (one of HVA types)
• Involved in dendritic calcium spikes
• Blocked by dihydropyridines
• Nifedipine, nimodipine
• Drugs used to treat heart conditions
• Decrease in calcium decreases cardiac
  contractions
• Open state prolonged by BayK8644

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Calcium Channels

• T type channels
• Transient (inactivation is prominent)
• Low voltage threshold (-65 mV, LVA type)
• Blocked by nickel, mibefradil
• Generates rhythmic bursts of action potentials
• Slow calcium spikes
• Burst of sodium spikes on top of calcium spike

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Calcium channels

• N type channel
• Neither transient nor long lasting
• Some inactivation
• Threshold between T type and L type (-20 mV)
• Another HVA type of VDCC
• Blocked by w-conotoxin GVIA (cone snails)
• Participates in calcium dependent release of
  neurotransmitter in some cells

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Calcium Channels

• P type channel
• Found in cerebellar Purkinje cells
• Blocked by w-agatoxin IVA (Funnel web spider)
• Generates dendrite calcium spikes
• Similar to L type channel
• No inactivation
• Threshold -20 mV

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Calcium Channels

• Q type channel
• Similar to P type
• Sometimes referred to collectively as P/Q type if
  only channel kinetics, voltage dependence
  assessed
• Blocked by w-conotoxin MVIIC
• R type channel
• Residual - remains after all else blocked!
• Recently discovered channel blocker SNX-482

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Calcium channels

• Structure
• Large ?1 subunit
• Sufficient to form channel
• 175-230 kDa
• One or more smaller subunits
• ?, ?, ?, ?2
• Modulates channel kinetics
• Multiple distinct ? and ? genes
• All can have alternative splicing

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Not all K channels subunits have 6 transmembrane
domains
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Hyperpolarization Activated

• Activates below -60 mV
• One type is permeable to Na and K
• ER -30 mV
• Inward current at resting potential
• Causes slow depolarization
• Rate of activation modified by neurotransmitters
• Channel gating is modulated by cAMP and cGMP
• Also called HCN channel has 6 TM segments per
  domain
• Another type permeable to K ER -80 mV
• Discussed with potassium channels

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Cardiac "Funny" Channel

• First identified Hyperpolarization activated
• Inward IF current causes slow depolarization
• Depolarization causes AP in cardiac cells
• AP deactivates IF
• AHP re-activates IF current
• Cycle begins again
• Pacemaker

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Cardiac "Funny" Channel

• Rate of IF activation influences rate of
  depolarization
• Faster activation faster depolarization
• Faster depolarization more frequent action
  potentials
• Rate of activation modulated by
• Norepinephrine (increases rate)
• Acetylcholine (decreases rate)

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Thalamic IH

• Depolarization produces bursts in Thalamus
• IH causes depolarization
• Depolarization activates IT (ICaT)
• IT produces calcium spike
• Calcium spike
• activates sodium spikes
• deactivates IH
• Inactivates IT

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Thalamic IH

• AHP repolarizes membrane
• IH activates
• IT de-inactivates
• cycle repeats
• Neuromodulators control rate of oscillations
• Wakefulness (Norepinephrine) produces persistent
  depolarization and sodium spikes
• IH deactivated
• IT inactivated

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Potassium Channels

• Most diverse group of channels
• Voltage dependent
• Some with inactivation
• Calcium dependent
• Hyperpolarization activated
• Some modulated by G proteins
• Leak channels
• Both functionally and structurally diverse

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Leak channels
Calcium dependent
Inward rectifiers
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Auxillary Subunits of K channels

• Kv family
• Beta subunits confer rapid inactivation,
  influence membrane targeting
• Caspr2 plays role in membrane targeting
• Slowpoke family
• Beta subunits modulate voltage and calcium
  dependence
• Slob and Slip modulate voltage and calcium
  dependence, influence membrane targeting

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Shaker Family

• Kv1.1 through Kv1.8
• Blocked by TEA (non-specific)
• Also blocks BK and KA to some extent
• Several subunits blocked by dendrotoxin
• From mamba snake venom
• Role
• Spike repolarization
• Spike timing

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KCNQ family

• Similar to shaker but with long carboxyl tail
• Subunits KCNQ2 (kv7.2) KCNQ5 (kv7.5) in CNS
• Slowly activating and non-inactivating
• Inhibited by G protein coupled receptors
• M current Acetylcholine (Muscarine) modulated
• PIP2 required for channel opening
• ACh stimulation causes PIP2 hydrolysis and
  depletion
• Plays a role in familial epilepsy

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Calcium Dependent Potassium Channels

• Activated by calcium
• Some are voltage sensitive, also
• Blocked by
• Calcium buffers BAPTA, EGTA
• Calcium channel blockers cadmium, cobolt
• Barium
• Flows through calcium channels, but can't bind to
  KCa channels
• Specific Apamin (SK), Iberiotoxin, charybdotoxin

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Calcium Dependent Potassium

• Function
• Control frequency of repetitive firing
• Spike frequency adaptation
• Repolarization of action potential
• Function depends on kinetics of activation
• Slow activation produces spike frequency
  adaptation
• Fast activation produces large AHP

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Calcium Dependent Potassium

• 6 transmembrane domains
• S0 seventh membrane segment that puts amino
  terminal extracellullar

• Long carboxyl terminal
• 2/3 of protein
• Contains calcium sensitive sequences

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Calcium Dependent Potassium Channel

• Channel is tetramer
• mRNA has alternative splicing
• Two main types
• Large conductance
• Maxi K or BK (IC)
• Requires voltage for activation
• Slowpoke locus
• Two subfamilies
• Fast and slow kinetics

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Calcium Dependent Potassium Channel

• Two main types
• Small conductance
• SK (IAHP)
• Voltage independent
• SK locus
• Four subfamilies
• Other types
• Intermediate conductance, gene unknown

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Transient Potassium Channels

• Also called A or KA current (ID in hippocampus)
• Rapid activation (ms)
• Threshold near -40 mV
• Inactivation
• Complete at -40 mV
• Need to hyperpolarize cell to see current
• Minimal current active at resting potential

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Transient Potassium Current

• Function
• Slows Firing
• Inactivated by AP
• De-inactivated by AHP
• Activates as VM returns to spike threshold
• Slows return to threshold
• Allows arbitrarily low firing frequencies
• Increases latency to spike in neurons with
  hyperpolarized resting potential
• Depolarization activates A current
• No spike occurs until current has inactivated

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Transient Potassium Channel

• Multiple types
• Fast and slow types in spiny projection neurons
• Kv1.4, Kv4.1, Kv4.2
• Blocked by 4-aminopyridine (4AP)
• Not specific to KA, low conc. blocks some other
  Kv channels

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Inward Rectifier Potassium

• Two main types
• Inward Rectifier
• KIR subunits
• Pore domain similar to Shaker
• Only two membrane spanning regions (S5 and S6)
• P domain connects S5 and S6

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Inward Rectifier Potassium

• Two main types
• G protein activated inward rectifier
• Kir type 3 subunits
• Activity regulated by G protein coupled receptors
• Activated by Gb(1-4)g subunits
• Inhibited by Gb5g or Gaq/11 subunits
• Gaq/11 inhibition due to PIP2 hydrolysis and
  depletion (similar to KCNQ)

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Leak Potassium Currents

• Active at resting potential
• Major contributor to resting membrane potential
  in some neurons
• Not voltage dependent
• Neuromodulators decrease current causing
  depolarization
• Decrease outward conductance appears as inward
  current
• Gas anesthetics open these channels
• Four transmembrane domains
• TWIK, TASK, TREK

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