Neuropharm Exam 2

K+ Channel Functions

• Play important roles in determining neuronal excitability

• Shape action potential

• Set resting potential

• Contribute to excitability of dendrites and cell body, as well as axon

Neuronal excitability

Property or characteristic(s) of neurons that dictate how easy or likely it is for a neuron to fire an action potential.

Contributors:

• Sensitivity to neurotransmitters

• Difference between membrane potential and threshold

Neuron Summation

• Threshold depolarization must be reached for AP to fire

• Whether threshold is reached is determined by:

  • Number & size of excitatory inputs

  • Number and size of inhibitory inputs

  • Difference between resting pot’l and threshold

Post-synaptic potentials

6TM K+ Channels

– Voltage gated K+ channels

– Ca2+ activated

2TM K+ Channels

– Inward Rectifying

– Includes K_ATP Channels

K+ Channel Classes

• K+ channels can also be divided into fast vs slow opening channels

• Some VGK+C will open quickly in response to depolarization

• Others are delayed → for example, the VGK+C that mediates the falling phase of the AP

Fast Opening Channels (KA)

Blocked by 4-AP

Kv Family

• Delayed rectifiers: members of K_v1-3, 7, 10 families

  • Shape repolarization and hyperpolarizaton after action potential → I_K(V)

  • Localize to axons → blocked by TEA

A-type channels (Kv4 family)

• Open briefly in response to depolarization after period of hyperpolarization → fast opening and inactivating

• Regulate action potential frequency (think of summation)

• Blocked by 4-AP

Ca2+ Gated K+ Channels (KCa Family)

• Two types → Large (B_K) and small (S_K) conductance channels

• Regulate rate of action potential firing

Bk Channels

• These channels mediate long after hyperpolarization (AHP),

• Increase interspike interval (decrease AP firing rate)

• Also regulate repolarization in dendrites

Sk Channels

• These channels mediate long after hyperpolarization (AHP)

• Regulate rate of action potential firing, shape EPSP

• Blocked by bee toxin apamin (small peptide, crosses BBB)

KIR Structure

Inward Rectifying K+ Channels (KIR)

• Establish resting membrane potential

• Inactivate when membrane depolarizes

• Blocked by TEA, spermine

KIR3 Family (GIRKs)

K_IR regulated by metabotropic neurotransmitter receptors (muscarinic acetylcholine, GABAB…)

Kir Block

• Block by Endogenous Polyamines

• Measure current through Kir channels

• Inward rectification of Kir2.1 current in cell-attached patch vs inside-out patch.

Spermine biding

Binds acidic residues in Kir pore to block it

KATP Channels

• Consist of KIR subunits (pore forming) and sulfonylurea (SUR) subunits

• Open in response to low cellular ATP, lowers membrane potential (hyperpolarizes)

• Protect neurons by reducing activity when energy reserves are low

KATP Channel Antagonists

In periphery → drugs that bind SUR subunit and reduce KATP channel opening increase insulin secretion, treating Type II diabetes and hypoglycemia.

Includes Glibenclamide and Repaglinide.

KATP Channel Openers

• Can be neuroprotective (Iptakalim?)

• Used as antihypertensives as they relax smooth muscle of vasculature

• May also treat hair loss

VGIC Structures

VGCC Subunits

VGCC Localization and Sensitivity

VGCC Types

Dihydropyridines

• Large family of L type channel blockers

• Some are clinically used antihypertensives

Phenylalkylamines

• L type channel blockers

• Some are clinically used antihypertensives

• Verapamil may also treat cluster headache

w-Conotoxins

• Cone snails use small peptide toxins to paralyze their prey

• This toxin’s GVIA blocks N VGCCs

• N type channels are involved in pain sensation → w-Ctx is analgesic (intrathecal)

  • 1000X more potent than morphine

w-Agatoxin

Small peptide P type channel blocker

T Type Channels

• So called “transient” channels as they open at small depolarization and inactivate quickly upon further depolarization

• Set “pacemaker” current in thalamic neurons

• Block of thesechannels (Cav3.1-3) by ethosuximide prevents absence seizures

Ca2+ in Neurons

• Normally, intracellular [Ca2+] ~10-100nM, extracellular is ~1-3mM

• Huge gradient → massive influx if channels open

• Strong depolarization

• Second messenger → many proteins are regulated by Ca2+

Ca2+ signaling

Ca2+ Binding Domains

• Many proteins sense calcium through Ca2+ binding domains

• Calcium binding induces conformational change

• Change activity of channel/enzyme

• Change in protein-protein interactions

• C2 domain, EF Hands

EF Hand Domain

Common Ca2+ domain found in many Ca2+ sensing proteins

Example protein → Calmodulin

C2 Domains

• b-sheet Ca2+ binding domain

• Synaptotagmin → Ca2+ sensor for neurotransmitter release

Presynaptic Ca2+ Regulation

• VG Ca2+C are near active zone in neurons of CNS

• Induce large local rise in Ca2+ concentration

• Ca2+ is quickly removed into intracellular buffers like ER, or pumped into extracellular space

Vesicular neurotransmitter release

• Docking → vesicle is brought close to target membrane

• Priming → molecular machinery for fusion is “primed”

• Fusion → upon Ca2+ influx, vesicle fuses

• Endocytosis → vesicle is retrieved

• Recycling → vesicle is refilled with neurotransmitter

Vesicle Proteins

SNAREs

• On vesicular membrane (v-SNAREs)

• On target membrane (t-SNAREs)

• Bring 2 membranes together to fuse

Synaptotagmin

Ca2+ sensor for vesicular neurotransmitter release

Fusing Proteins

a-Latrotoxin

Forms Ca2+ Channel, Inducing Massive Exocytosis

Botulinum Toxins

• Initially taken into cells by endocytosis, then exit vesicles into cytoplasm

• These toxins catalyze proteolysis (cleavage) of SNARE complex

• Prevents vesicular neurotransmitter release, at NMJ this causes paralysis

• Highly toxic → estimated human median lethal dose (LD-50) of 1.3–2.1 ng/kg intravenously or intramuscularly and 10–13 ng/kg when inhaled

NT Release Disruption

Botulinum toxin uses (Botox)

• Strabismus (eye muscle disorder)

• Chronic migraine

• Hyperhidrosis (excess sweating)

• Cosmetic applications, especially wrinkle reduction

Cholinergic projections

Acetylcholine

• In CNS it modulates → sleep, attention, memory/cognition, and pleasure/reward

• In PNS

  • Excitatory NT of neuromuscular junction (nicotinic)

  • Several autonomic roles (especially parasympathetic)

Acetylcholine Synthesis

• Acetate (from acetyl-CoA) plus choline = acetylcholine

• You need to be able to draw acetylcholine

Acetylcholine Metabolism

1. Acetylcholine is synthesized in the cytosol of presynaptic terminal by choline acetyltransferase

2. Loaded into vesicles by a transporter, VAChT

3. Released into synaptic cleft

4. Broken down into acetate and choline by acetylcholinesterase

5. Choline is recycled by Na+/Choline transporter

Vesamicol

Vesicular acetylcholine transporter (VAChT) inhibitor

Hemicholinium

Choline transporter (ChT) inhibitor

nAChR Channel structure

• One receptor binds at least 2 molecules of ACh to open

• nAChRs have hydrophobic ring in pore

• Leucine residue highly conserved in M2 pore lining helix

• Forces cations to shed hydration before entering cell

Homomeric vs Heteromeric

nAChR Channel Properties

• Cation channels that pass Na+, K+ → some may pass Ca2+

• nAChRs desensitize → pass less current after prolonged ligand binding

• Intrinsic property of nAChRs

• Can be modulated by posttranslational modifications to channel

Nicotine

• Acetylcholine Receptor Agonist

• Tobacco was brought to new world by Spanish & Portuguese

• Popularized by French ambassador to Portugal, Jean Nicot

nAChR Agonists

• Acetylcholine

• Nicotine

• Arecoline (areca/betel nut) → (also muscarinic agonist)

• Coniine (poison hemlock)

• Carbachol → (also agonist for mAChR)

Turbocurarine (curarine)

• Active component of curare

• Competitive, reversible antagonist of nAChR

• Harmless if ingested orally (excellent hunting poison)

Varenicline

partial agonist at nAChR

Mecamylmine

Noncompetitive nAChR antagonist → Also an antihypertensive

a-Bungarotoxin

• 74 amino acid peptide

• Competitive, irreversible antagonist of nAChR

• Can be used to label (microscopy) and purify nAChR

M1,3,5

Gaq coupled receptors → Have excitatory effect on postsynaptic cell

M2,4

Gai coupled receptors → Have inhibitory effect on postsynaptic cell

Ga proteins

Acetylcholine Autonomic

• Parasympathetic → “rest and digest”

• Cardiac muscle expresses M2 muscarinic receptors

  • Inhibitory receptors → slows down heart

• Smooth muscle and secretory organs express M3 muscarinic receptors

  • Increase smooth muscle contraction and secretion

Muscarine

• Nonspecific agonist of mAChRs

• Symptoms → Salivation, Sweating, Tearing, Diarrhea

• Death by cardiac/respiratory failure

• Antidote → atropine

Muscarinic Agonists

• Muscarine

• Arecoline (areca/betel nut) → (also nicotinic agonist)

• Oxotremorine

• Carbachol → (also nicotinic agonist)

• Pilocarpine

Organophosphates

• Acetylcholinesterase Inhibitors

• Tabun & sarin are volatile (gas) nerve agents

• Diazinon is a ____ insecticide

Carbamates

Reversible, covalent inhibitors of acetylcholinesterase (AChE)

acetylcholinesterase inhibitors in CNS

Uses include:

– Learning deficits associated w/neurodegeneration (Alzheimer’s)

– Cognitive impairment associated with schizophrenia

– Tacrine is no longer available owing to liver toxicity

acetylcholinesterase inhibitors in PNS

• Myasthenia Gravis → loss of nAchRs at NMJ

• Decreasing AchE activity helps compensate

Physostigmine

• Acetylcholinesterase Inhibitor

• Natural product from Calabar bean

• Used medicinally, along with 2 semi-synthetic derivatives

Rivastigmine

• Acetylcholinesterase Inhibitor

• Used for CNS applications, such as treating dementia

Neostigmine

• Acetylcholinesterase Inhibitor

• Cannot cross into CNS, used for PNS applications

Nicotinic agonists

Act at neuromuscular junction to increase muscle tone/induce paralysis

Muscarinic agonists

Increase activity of parasympathetic nervous system

• Secretory responses of the such as salivation and lacrimation (tearing).

Muscarinic antagonists

Inhibit the actions of the parasympathetic system → para-sympatholytic agents

SLUDGE

• Symptoms of increased Ach activity in parasympathetic nervous system (mAChRs)

• AChEI poisoning → possible nAChR symptoms plus:

• Salivation

• Lacrimation (tearing)

• Urination

• Diaphoresis (sweating)

• Gastrointestinal mobility (diarrhea)

• Emesis (vomiting)

Atropine

• Nonspecific, competitive mAchR antagonist

• Counteracts bradycardia

• Counteracts muscarine and AchEI poisoning

• Reduces salivation, diarrhea

• Causes hallucinations at high doses

Benzatropine

• Nonspecific competitive mAchR antagonist

• Reduces parkinsonism, tremor

Glutamate

• Major fast, excitatory NT of CNS

• Non-essential amino acid

• Does not cross blood brain barrier → Synthesized from precursors in brain

• 3 types of receptors

  • AMPA/Kainate

  • NMDA

  • Metabotropic

Glutamate Synapse

Glutamate Reuptake

• Glutamine is metabolized into glutamate by glutaminase

• Glutamate is removed from synaptic cleft by neurons and astrocytes

• Astrocytes metabolize glutamate into glutamine, release glutamine, neurons take up glutamine and convert it back to glutamate

Glutamatergic signaling

• Short term excitation → postsynaptic depolarization

• Longer term signaling → synaptic plasticity

• Excess glutamate → excitotoxicity

AMPAR

• Ionotropic glutamate receptors

• Mediate the majority of excitatory neurotransmission in the CNS

• Tetramers of GluA1-4

  • Dimer of dimers → GluA1/2 or 2/3 most common

  • Homotetramers of GluA1 rare but exist

• Ligand gated cation channels (usually Na+, sometimes Ca2+)

  • Ca2+ permeability depends on Q/R editing site of GluA2 subunit

GluA2 Q/R Editing

• The mRNA GluA2 subunit of AMPARs is edited to replace a codon for glutamine (Q) to arginine (R)

• This Q to R substitution makes AMPARs with GluA2 impermeable to calcium

• This editing takes place in the vast majority of GluA2 mRNAs, meaning GluA2 containing AMPARs do not pass Ca2+

AMPAR Desensitization

AMPARs desensitize in presence of prolonged agonist

AMPAR Agonists

Research tools:

2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propanoic acid (AMPA)

Quisqualate (also Group I mGluR agonist)

(S)-(-)-5-Fluorowillardiine

AMPAR Competitive Antagonists

• Quinoxaline family

• Usually abbreviated with “QX” at end

• The most important of these is NBQX

• Research tools

AMPAR Noncompetitive Antagonists

• 2,3-benzodiazepines

• Anticonvulsants

• Neuroprotective (reduces excitatory activity)

• Tofisopram is available in Europe, not approved in US or Canada- CYP3A4 inhibition?

Voltage-dependent Blockers

• Bind GluA2-lacking AMPARs

  • Or unedited GluA2 with Q residue

• Block AMPAR-mediated Ca2+ current

• Endogenous polyamine confers inward rectification

Cyclothiazide

• AMPARs desensitize in presence of prolonged agonist

• Desensitization can be reduced/blocked by _____

• Positive allosteric modulator

Nootropics

• Substances that improve cognition, concentration, memory

• Ones we’ve seen before → cholinergic agents

• Some AMPA modulators (ampakines) are ____ → Positive allosteric modulator

Ampakines

• Racetams (some, not all) → Aniracetam and Piracetam (other CNS effects)

• Benzylpiperidines: AMPAR-specific ampakines

NMDA Receptor

• Tetrameric channels of GluN1-3 (old nomenclature: NR1-3)

• GluN1 is obligatory subunitt → GluN1/2 most common

• Similar topology to AMPARs

• Ligand gated cation channels (Na+, Ca2+)

• Also voltage gated (kind of)

Glutamate EPSCs

Glutamate/Glycine Co-agonists

Glutamate and glycine are NMDAR coagonists

Mg2+ NMDAR Block

Required for voltage dependent block of NMDARs at resting/hyperpolarized potentials → so pass current only when postsynaptic membrane is depolarized

NMDAR’s Asn residues in pore may confer Mg2+ block

NMDAR coincidence detectors

• Presynaptic glutamate release

• Postsynaptic depolarization

N-methyl-D-aspartic acid

Partial NMDAR agonist for the glutamate site

Cycloserine

Partial NMDAR agonist for the glycine site

May be useful adjunct therapy for schizophrenia