Transcript calcium channel blockers

CALCIUM CHANNEL
BLOCKERS
PHRM-520-L.S.No-7th-E-01
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Calcium Flow Pathways
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+2
Ca ION
• Calcium ions are the principal intracellular
signaling ions (Release of stored Ca+2)
• Regulate excitation–contraction coupling
secretion
• Activity of many enzymes
• Excitatory Neurotransmitter
• Ion channels, Hormone
• Transporters such as the sodium-calcium
exchanger (NCX), also play important
roles in [Ca2+] regulation.
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Types of Muscle Tissue
Skeletal
•Attach to and move skeleton
•40% of body weight
•Fibers = multinucleate cells (embryonic
cells fuse)
•Cells with obvious striations
•Contractions are voluntary
Cardiac: only in the wall
of the heart
•Cells are striated
•Contractions are
involuntary (not
voluntary)
Smooth: walls of hollow organs
•Lack striations
•Contractions are involuntary (not voluntary)
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Similarities…
• Their cells are called fibers because
they are elongated
• Contraction depends on myofilaments
–Actin
–Myosin
• Plasma membrane is called
sarcolemma
–Sarcos = flesh
–Lemma = sheath
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[Ca2+]i Three Best Studied Roles:
1. Contraction of Muscle
2. Secretion
3. Gating
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The Ion… Ca2+
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Sodium-Calcium Exchanger (NCX)
• The calcium that enters the cell during action
potentials must be removed from the cell
otherwise an accumulation of calcium would lead
to cellular dysfunction.
• Calcium is removed from cells by two basic
mechanisms.
• 1. An ATP-dependent Ca++ pump that actively
removes calcium from the cell.
• 2. Sodium-calcium exchanger.
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When the cell is depolarized and has a positive membrane
potential, the exchanger works in the opposite direction (i.e.,
Na+ leaves and Ca++ enters the cell).
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Drugs Acting on Calcium Channels
Calcium Channel Blockers
L-type Ca channel
•Verapamil
blocker
NPQR Ca channel
•Gabapentin
blocker
T-type Ca channel
•Pimozid
blocker
•Diltiazem
•Mibefradil
•Nifedipine
•Succinimide
Antiepliptic drugs
•Phenytoin
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Cardiac-Types of Ca+2 Channels:
2 types of Ca2+ channels:
– L- (low threshold type)
– T-type (transient-type)
Transport Ca2+ into the cells
The L-type channel is found in all
cardiac cell types and vascular smooth
muscle.
The T-type channel is found:
pacemaker, atrial, and Purkinje cells.
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Difference Between L/T Type
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Excitation-contraction (E-C) coupling
• It is the process depolarization of the
muscle fiber membrane, elicited by a
nerve action potential, triggers the
release of Ca2+ from the sarcoplasmic
reticulum (SR).
• resulting rise in intracellular Ca2+
• concentration activates the troponin
complex,
• initiating the contraction of the muscle.
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Myofibrils
• Made of three types of filaments (or
myofilaments):
– Thick (myosin)
– Thin (actin)
– Elastic (titin)
titin_____
______actin
_____________myosin
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Contraction
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Myosins belong to a large superfamily
Motor proteins that move along actin
filaments, by hydrolyzing ATP.
There are 20 classes of myosin
Distinguished on the basis of the
sequence of amino acids in their ATPhydrolyzing motor domains.
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Myosin II
-helical
coiled coil
light
chains
heavy
chain
motor
domains
Myosin II first studied for its role in muscle
contraction, but it functions also in non-muscle
cells.
Myosin II includes 2 heavy chains.
 The globular motor domain of each heavy
chain catalyzes ATP hydrolysis, and
interacts with actin.
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2 Light chains, designated
essential & regulatory,
wrap around the neck
region of each myosin II
heavy chain.
light chains may help to
stiffen the neck.
Myosin
head & neck
PDB 2MYS
PDB 1CDM
Ca++- Calmodulin wrapped
around its target peptide
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• Each heavy chain continues into a tail
domain in which heptad repeat sequences
promote dimerization by interacting to form
a rod-like -helical coiled coil.
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Another
Picture
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Special Functional Characteristics of Muscle
 Contractility
 Only one action: to shorten
 Shortening generates pulling force
 Excitability
 Nerve fibers cause electrical impulse to travel
 Extensibility
 Stretch with contraction of an opposing muscle
 Elasticity
 Recoils passively after being stretched
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Sliding Filament Model
__relaxed sarcomere__
fully contracted
Sarcomere
shortens because
actin pulled
towards its middle
by myosin cross
bridges
Titin resists overstretching
_partly contracted_
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CALCIUM CHANNEL
• Calcium is stored in the sarcoplasmic
reticulum
• When the impulse is initiated the T tubules
release free Ca++
• This free Ca++ reacts with the troponin to
increase the number of cross bridges
(actin + myocin)
• Just as increasing the number of persons
pulling on a rope in a “tug of war” will
increase the tension or the pull on the
rope
• This increase in the number of active
cross bridges will increase the strength30of
the cardiac contraction
High resolution
electron microscopy
has detected
conformations
consistent with the
hand-over-hand
stepping mechanism.
processive movement of
myosin V along F-actin
Animation: Myosin V walking along an actin
filament.
Based on electron microscopic images of myosin V fragments (part
of the tail domain with 2 heads) attached to actin filaments in what
is interpreted as different stages of the reaction cycle.
(By M. L. Walker, S. A. Burgess, J. R. Sellers, F. Wang, J. A.
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Hammer, J. Trinick & P. J. Knight.)
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Structure of Ca+2 Channel
• A combination of 5 subunits, α1, α2, β, γ, and δ, unite to form
the channel in its native state.
• The β subunit increases channel expression ≈10-fold
and accelerates the activation and inactivation kinetics.
• The α1c subunit, Cav1.2, is the cardiac-specific subunit
http://calcium.ion.ucl.ac.uk/calcium-channels.html
http://www.sigmaaldrich.com
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Ca+2 Channel α1c subunit
• 4 homologous domains
• Each domain consists 6 membrane-spanning segments.
• The P-loop of each domain contributes a glutamate
residue (E) to the pore structure. pore loop contributes
to selectivity
• These residues (EEEE) are critical for calcium selectivity;
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The α1c subunit, Cav1.2, is the cardiac-specific subunit
Alpha-1 Subunit Structure
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http://calcium.ion.ucl.ac.uk/calcium-channels.html
Ribbon Structure of Alpha-1
http://calcium.ion.ucl.ac.uk/calcium-channels.html
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Structure/Function
• Positively charged lysine and arginine
residues in the S4 transmembrane
segment thought to form the voltage
sensor
• The carboxyl terminus has multiple Ca2+
binding sites and Ca-calmodulin–
dependent kinase activity.
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Two Primary Proteins
• Involved in the initial events of EC coupling:
–1. Dihydropyridine receptor (DHPR)
–2. Ryanodine receptor (RYR),
• are both Ca2+ channels.
• Skeletal and cardiac muscle have
• different isoforms of both the
DHPR and RYR.
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DHPR in Skeletal Muscle
•
•
•
•
•
•
•
It is an L-type Ca 2+ channel,
is composed of four subunits:
α1S(190–212 kDa),
α2–
β(52–58 kDa),
γ (25 kDa).
δ(125 kDa),
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DHPR in heart
• The cardiac DHPR has three known
subunits:
• α1C (240 kDa)
• α2–δ(125 kDa)
• β(62 kDa).
• The γ-subunit has not yet been
identified as a subunit of the cardiac
channel.
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α1-Subunit of the DHPR
• α1-subunit of the DHPR forms
the channel pore
• and contains the binding sites
for channel-specific drugs
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Excitation-contraction (E-C) coupling:
• Depolarization of the muscle fiber
membrane by a nerve action potential,
• triggers the release of Ca2 from the
sarcoplasmic reticulum (SR)
• resulting rise in intracellular Ca2+
• concentration activates the troponin
complex
• initiating the contraction of the muscle.
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Cardiac Muscle (CIC)
• In cardiac muscle the mechanism
• of E-C coupling involve Ca2+
induced Ca2+release(CICR).
• The cardiac DHPR serves as a
functional voltage-dependent Ca2+
channel allowing entry of
extracellular Ca2+which raises the
local intracellular Ca2+concentration
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Calcium Induced Calcium Release (CICR)
1
2
3
Intracellular [Ca]
10-7 to 10-5 M
1. Ca++ enters the cell through L-type calcium channels
2. Ca++ stimulates Ca++ release from the SR via RyR
3. Ca++ interacts with contractile proteins to initiate
shortening of the myocyte
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Cardiac Muscle (CIC)
• Release of Ca2+ from the SR is
controlled by the Ca2+ release
channel or RYR.
• The RYR1 and RYR2 are
homotetramers
• with a subunit molecular mass of
~565 kDa, and they share 66%
sequence identity and ~80% overall
homology
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• Approximately 4/5 of the RYRs
are predicted to be cytoplasmic,
with
• only 1/5 of the molecule at the
carboxy terminus forming the
• luminal and membrane-spanning
domains
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CALIUM CHANNEL BLOCKERS
THREE CLASSIFICATIONS:
PHENYLAKYLAMINES
1,4-DIHYDROPYRIDINES
BENZOTHIAZEPINES
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Ca+2 Channel Blockers
• The Three classifications differ in their Tissue
Selectivity,
• Their Binding Site, location with the Alph 1
Subunit,
• Their mechanisms of calcium blockade
• 1,4 dihydopyrimidines are selective for the
arteriolar beds
• The phenylalkylamines and
benzothiazepines are selective for the AV
node
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CALCIUM CHANNEL BLOCKERS
• BLOCK Ca+2 entry into
• Cardiac and vascular smooth muscle at
• The Alpha-1 subunit of the L-type voltage
gated Ca+2 ion Channel (slow channels)
• Reduce myocardial O2 demand by
decreasing cardiac afterload and augments
o2 supply by increasing blood flow
(coronaryvasodilatation)
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CALCIUM CHANNEL BLOCKERS
• Decreased myocardial contractility
• Decreased heart rate –decrease O2
demand (verapamil+diltiazam)
• Decreased activity of SA node
• Decreased rate of conduction of
cardiac impulses through the SA
node
• Vascular smooth muscle relaxation with
associated vasodilatation and decreases
in systemic blood pressure
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PHENYLALKYLAMINE
• Verapamil—primary site is the AV node,
used for angina, essential hypertension
• May be useful in maternal and fetal
tachydysrhythmias as well as premature
labor, however
• It may decrease uterine placental blood flow,
use caution
• Negative inotrope and chronotrope effects
may be enhanced with in beta antagonist
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• Used with caution with left ventricular
dysfunction, conduction abnormalities
or bradydysrhythmias, diltiazem better
tolerated
• Isoproterenol useful to increase hr in
drug induced heart block
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1,4-DIHYDROPYRIMIDINES
• Nifedipine—greater coronary and
peripheral arterial vasodilator properties
than verapamil
• The peripheral vasodilatation decreases
systemic blood pressure, this activates the
baroreceptors , increasing heart rate
• SL nifedipine has serious adverse side
effects; cerebrovascular ischemia,
myocardial ischemia, severe hypotension—
no longer used hypertensive emergencies54
NICARDIPINE
• Lacks affects on SA and AV node
• Has the Greatest vasodilating effects of
all the calcium channel blockers,
vasodilatation prominent in the
coronary arteries
• Used for acute hypertension
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• Nicardipine-lacks affects on the SA
and AV node, Minimal cardiac
depressant effects
• Greatest Coronary Arterial
Vasodilatation –no coronary steal
• Used in combination with Beta Blockers
for Angina
• 1,4-Dihydropyrimidines produce the
greatest dilatation of the peripheral
arterioles
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1,4-DIHYDROPYRIMIDINES
• Nicardipine• Available oral or IV route. Oral 1/3
life is 72 hrs.
• Metabolized by the liver and is 95%
protein bound
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1,4-DIHYDROPYRIMIDINES
• Oral Cardene
20 mg q8h
30 mg q8h
40 mg q8h
Dose Equivalent
I.V. Infusion Rate
0.5 mg/hr
1.2 mg/hr
2.2 mg/hr
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NICARDIPINE
• Dose not cause significant increase
in ICP
• CPP=MAP-ICP
• PRECISE CONTROL OF B/P WILL
MAINTAIN CPP
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NICARDIPINE
• Can be used as a tocolytic, with fewer
side effects, however pulmonary
edema has been reported
• Used to blunt the hemodynamic
effects of ECT
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NICARDIPINE
• Common side effects include; headache
(14.6%), N/V (4.9%) and tachycardia
(3.5%)
• Use caution with a patient in CHF
(Congestive Heart Failure) and is being
treated with a betablocker, advanced aortic
stenosis, significant left ventricular
dysfunction, portal hypertension, impaired
renal and hepatic function.
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NICARDIPINE
•
•
•
•
25mg/10cc
Must be diluted
25mg in 240cc = 0.1mg/cc
Premixed 20mg/200cc = 0.1mg/cc
Initiate 5mg/hr (50cc/hr)
• Do not exceed 15mg/hr (150cc/hr)
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1,4--DIHYDROPYRIMIDINES
• Nimodipine—vasodilating cerebral
arteries preventing or attenuating
cerebral vasospasm that
accompanies sub-arachnoid
hemorrhage
• Cerebral protection after global
ischemia as associated with cardiac
arrest
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BENOTHIAZEPINES-Diltazem
Blocks predominantly the
calcium
channels of the av node
Used for svt and essential
Hypertention
Minimal cardiodepressant effects
unlikely to interact with betaadrenergic drugs
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LOCAL ANESTHETIC
• VERAPIMIL HAS A POTENT LOCAL
ANESTHETIC ACTIVITY
• WHICH MAY INCREASE THE RISK
OF LOCAL ANESTHETIC TOXICITY
• WHEN REGIONAL ANESTHESIA IS
ADMINISTERED TO PATIENTS
BEING TREATED WITH THIS DRUG
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ANESTHETICS AND CA++
BLOCKERS
• BOTH ARE VASODILATORS MYOCARDIAL
DEPRESSANTS
• POTENTIATE BOTH DEPOLARIZNG AND
NONDEPOLARIZING NEUROMUSCULAR BLOCKING
AGENTS AND THE CIRCULATORY EFFECTS OF THE
VOLATILE AGENTS
• THERE IS NO EVIDENCE THAT PATIENTS BEING
TREATED CHRONICALLY WITH CA++ BLOCKERS
ARE AT INCREASED RISK FOR ANESTHESIA
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DANTROLENE
• DANTROLENE AND VERAPAMIL OR
(DILTIAZEM) CONCURRENTLY,
RESULTS IN EXTREME
HYPERKALEMIA—NEED HEMODYNAMIC
MONITORING
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CALCIUM CHANNEL
BLOCKERS
• MAY INTERFER WITH PLATELET
FUNCTION
• MAY INCREASE PLASMA
CONCENTRATION OF DIGOXIN;
DECREASING PLASMA
CLEARANCE
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CALCIUM CHANNEL
BLOCKERS
• RISK OF CHRONIC TREATMENT
• INCREASED BLEEDING WITH
DIHYDROPYRIMIDINE DERVATIVES
• INCREASE RISK OF CANCER
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Cardiac Muscle (CIC)
• In cardiac muscle the mechanism
of E-C coupling involve Ca2+
induced Ca2+ release(CICR).
• The cardiac DHPR serves as a
functional voltage-dependent Ca2+
channel allowing entry of
extracellular Ca2+which raises the
local intracellular Ca2+concentration.
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Proteins Bound to DHPR or RYR
• In addition to (mechanical gating or
CICR),
• Ca2+ release is likely to be
modulated by other proteins bound
to the DHPR or to RYR.
• One of these protein is Calmodulin
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Mutation
Timothy syndrome is a multi-system
disease e.g. cognitive abnormalities,
immune deficiency, hypoglycemia, as
the result of mutations of CaV1.2.
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The mutation of glycine to arginine:
converts a neighboring serine to a
consensus site for phosphorylation by
calmodulin kinase.
The phosphorylation of this site
promotes a slow gating mode of the
calcium channel, increasing Ca2+ entry
and resulting in cytotoxicity.
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Class IV drugs
• The Ca2+ channel is the target for the
interaction with class IV
antiarrhythmic drugs.
• Phenylalkylamine, verapamil and the
benzothiazepine, diltiazem.
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K-ION CHANNEL
• Crucial Regulator Membrane
excitability
• CNS
• HEART
• Tissue
• Smooth muscle
• White blood cells
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Drugs acting on potassium
Channels
Potassium Channel Blockers
Anti-arrhythmic
Hypoglycemic
•Sotalol
•Bretylium
•Sulfonyl-Urea Agents
Not selective to K
•Amiodarone
Difetilide, Ibutilide Highly
selective to K
•Non-Sulfonyl-Urea
Agents
•Repaglinide
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Antiarrythmic IV -LIKE
• K channel opener (hyperpolarization)
• Repolarization: Unchanged
• Example: Adenosine
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Transmembrane segments (S4)
senses voltage shifts in the
Membrane Opens the ion channel
KV family
EAG family
KCNQ family
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•
•
•
•
Each family contain a number of members:
They are known as subfamily
Each of this is the product of different gene
KV1.5 most important sub-family member in
the human heart
• This channel makes the ultra rapidly
activating
• Delayed in the mammalian atrium.
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CHLORIDE CHANNEL
Ligan Gated
Benzodiazepine
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The GABA-A Receptor ?
• Major mammalian inhibitory neurotransmitter
receptor
• Pentameric integral membrane protein
containing an ion channel selective for
chloride ions.
Extracellular part
Transmembrane part
Neuroreceptors
• Activation causes a net change in the electrical
properties (membrane potential) of that neuron
and determines its activity.
• Increase in Chloride ions =
HYPERPOLARIZATION
• Neurons less likely to fire.
• Calming, tranquilizing, prevents us being
overwhelmed by stressful situations!
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Benzodiazepine Binding Site
• Allosterically stimulate the function of the
GABA-A receptor.
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Allosteric stimulation
• Binds at its own separate site away from the
active site.
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GABAA receptor
• Benzodiazepine receptors associated
with GABA chloride channel
complex
• GABA agonists cause opening of the
Cl channel.
• benzodiazepine receptor is a
modulating unit, modifying the
response to GABA.
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Ligand-gated ion channel neuroreceptors
Channel closed
ClClCl-
Cl-
ClNT neurotransmitter
Cl-
Cl-
Cell membrane
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Ligand-gated ion channel neuroreceptors
Cl-
ClCl-
pore
Cl-
Cl-
Cl-
Cl-
ClCl-
ClNT
ClCl-
Cl-
ClCl-
Cl-
Cl-
Cl-
Cl90
Ligand-gated ion channel neuroreceptors
NT
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Ligand-gated ion channel neuroreceptors
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Na+ Channel Modulation
• Phosphorylation
• sodium channel function is modulated by serine/threonine
and tyrosine kinases as well as tyrosine phosphatases (Yu
et al, Science 1997)
• Mutation – altered amino acid sequence/structure can
change the biophysical properties of the Na+ channel
• Pharmacology – block Na+ channel to reduce the
conductance
• Proteolysis- (cleavage) Proteases may cleave specific
residues or sequences that inactivate a channel, or
significantly alter the biophysical properties
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Why Na+ Channels/Modulation Are
Important
•
•
•
•
•
Neuronal depolarization, Action Potential
Neuronal Excitability
Cardiac Excitability
Muscle Excitability
The basis of neuronal/cardiac/muscular function
relies on the propagation of action potentials,
down axons, sarcolemma, myocardium, as well as
requiring synaptic transmission.
• Differential excitability alters the electrical
conduction/transmission properties of the “circuit”
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Na + Channel Blockers/Pharmacological
Agents
•
•
•
•
•
•
•
Tetrodotoxin (TTX)
Amioderone (Antiarrhythmic)
Lidocaine (Anesthetic agents)
Procainamide (Anesthetic agents)
Mexilitine (Antiarrhythmic)
Ketamine (General Anesthetic agents)
Many, many others
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Some Na+ Channels Outside The
Nervous System
• Naf – “Funny Current” in pacemaker cells of the
heart (SA node/ectopic pacemakers)
• SA node –Beta 1 receptor
(sympathetic receptor)
• Nav in the myocardium, sarcolemma, and Ttubules and motor endplate
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Na+ Channel Activation
• Change in transmembrane potential results in a
conformation change in the Na+ channel
• The four S4 segment alpha helices translocate, thus leading
to the opening of the channel pore
• The energy of the conformational change in the channel
during activation is mediated by the reduction in overall
entropy of the system.
• The voltage sensor is a highly charged sequence of amino
acids that “aligns” itself according to the electrical field
present
• A change in transmembrane potential creates unfavorable
electrodynamic interaction for the voltage sensor, hence a
conformational shift lowers the energy of the system and
creates more favorable conditions
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Patch Clamping/Transfection
Transfection
1. Kv1.3 cDNA in Plasmid
2. Lipofectamine
complexing
3. Add to Dishes
4. Patch 28-48 hrs after
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Transition: A General Overview of
Articles Before Discussion
• From Basic structure/function relationships
to a gating mechanism
• The gating of a bacterial Na+ channel and
application of Na+ channel activation and
biophysical properties
• Article 1 – A gating hinge in Na+ channels:
a molecular switch for electrical signaling
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Conserved
glycine
In the S6 domain
Proposed conformational shift of A-helix caused by substitution of
Proline for G219
Prolines in alpha helices after the first turn (4th residue) cause a kink in
the helix.
This kink is caused by proline being unable to complete the
H-bonding chain of the helix and steric or rotamer effects that keep
proline from
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adapting the prefered helical geometry
Na+ Channel Gating
• Current theory holds that a change in
transmembrane potential “flips” the conformation
of the voltage sensor, thereby opening the channel
pore
• A mutation, G219P, glycine 219 changed to
proline alters the conformation of the S6 domain
• The mutant channel now favors a state much like
the “open” state of a wild-type channel
• NOTE: these bacterial Na+ channels are
homotetramers of identical subunits
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Regulation and Modulation in Na
Channels
Phosphorylation effects
•
• Mutations in ball-andchain affect inactivation
speed
• Cleavage of any part of
Na channel protein
• Drugs can be used as
modulators
• NO modulates Na
currents (Ribeiro et
al., 2007)
– NO donors reduce
peak Na current
• ENaC modulated by
accessory proteins
(Gormley et al., 2003)
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Na+ Channel Modulation
• Pharmacology – block Na+ channel
to reduce the conductance
• Proteolysis- (cleavage) Proteases
may cleave specific residues or
sequences that inactivate a
channel, or significantly alter the
biophysical properties
109
Na+ Channel Modulation
• Phosphorylation
• sodium channel function is modulated
by serine/threonine and tyrosine kinases
as well as tyrosine phosphatases (Yu et
al, Science 1997)
• Mutation – altered amino acid
sequence/structure can change the
biophysical properties of the Na+
channel
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Sodium Channels - Function
• Play a central role in the transmission of action
potentials along a nerve
• Can be in different functional states (3)
-A resting state when it can respond to a
depolarizing voltage changes
-Activated, when it allows flow of Na+ ions
through the
-Inactivated, when subjected to a “suprathreshold”
potential, the channel will not open
(hyperpolarization)
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Pharmacology (i.e. drugs of choice)
• Saxitoxin (STX), from red
tide, used to count Na
channels (Ritchie et al.
1976)
• Tetrodotoxin (TTX), from
fugu puffer fish, local
anesthetics also block Na
channel flux
– Local anesthetic: #
channels open at once
Saxitoxin
www.chemfinder.com112
• Single linked protein
makes up ion channel
– P-loop reflects speed
of inactivation
• ,  subunits modify
channel function but
are not essential to
create the pore
• Ligand-gated channels do
not have voltage sensor,
but ligand binding site
• Voltage gated channels
have voltage sensor on S4
in each domain
– Speculation: domain
sensors have special
functions (Kuhn and Greef,
1999)
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• Drugs bind to receptors
– Can be used to count receptors, block channels
(ex: identify which current is responsible for
some spiking)
• Na channel is not perfectly selective
– Also permeable to K+ ions, though much less
than Na+ (Chandler and Meves, 1965)
– Therefore, drug application may not necessarily
block one ion completely
• Drug responses are variable
– Cardiac cells respond less to TTX than skeletal
muscle cells (Ritchie and Rogart, 1977; Cohen
et al., 1981)
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