calcium channel blockers 1phrm-520-l.s.no-7th-e-01

CALCIUM CHANNEL

BLOCKERS

1PHRM-520-L.S.No-7th-E-01

Calcium Flow Pathways

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Ca+2 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. 3

Types of Muscle TissueSkeletal

•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) 4

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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 6

[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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• 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. 

Sodium-Calcium Exchanger (NCX)

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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 blocker

T-type Ca channel blocker•Verapamil

•Diltiazem

•Nifedipine

•Pimozid

•Mibefradil

•Succinimide Antiepliptic drugs

•Phenytoin

NPQR Ca channel blocker•Gabapentin

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. 16

Myofibrils

• Made of three types of filaments (or myofilaments):– Thick (myosin)– Thin (actin)– Elastic (titin)

______actin_____________myosintitin_____

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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 ATP-hydrolyzing motor domains.

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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.

-helical coiled coil

heavy chain motor domains

Myosin II

light chains

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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.

Ca++- Calmodulin wrapped around its target peptide

PDB 1CDM

Myosin head & neck

PDB 2MYS

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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__ _partly contracted_

fully contractedSarcomere shortens because actin pulled towards its middle by myosin cross bridges

Titin resists overstretching29

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 strength of the cardiac contraction

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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. Hammer, J. Trinick & P. J. Knight.)

processive movement of myosin V along F-actin

High resolution electron microscopy has detected conformations consistent with the hand-over-hand stepping mechanism.

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http://calcium.ion.ucl.ac.uk/calcium-channels.html http://www.sigmaaldrich.com

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

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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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Alpha-1 Subunit Structure

http://calcium.ion.ucl.ac.uk/calcium-channels.html

The α1c subunit, Cav1.2, is the cardiac-specific subunit

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Ribbon Structure of Alpha-1

http://calcium.ion.ucl.ac.uk/calcium-channels.html 37

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 E-

C 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. 39

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), 40

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. 43

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. 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

Intracellular [Ca] 10-7 to 10-5 M

1 2

3

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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 46

• 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 49

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 52

• 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 emergencies

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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 Dose Equivalent

I.V. Infusion Rate

20 mg q8h 0.5 mg/hr

30 mg q8h 1.2 mg/hr

40 mg q8h 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 64

BENOTHIAZEPINES-Diltazem

Blocks predominantly the calcium channels of the av node

Used for svt and essential

Hypertention

Minimal cardiodepressant effects unlikely to interact with beta-adrenergic 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 cells76

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Drugs acting on potassium Channels

Potassium Channel Blockers

Anti-arrhythmic Hypoglycemic

•Sotalol

•Bretylium

•Amiodarone

Difetilide, Ibutilide

Not selective to K

Highly selective to K

•Sulfonyl-Urea Agents

•Non-Sulfonyl-Urea Agents

•Repaglinide

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 CHANNELLigan 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

Cell membrane

neurotransmitterNT

Channel closed

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-Cl-

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Ligand-gated ion channel neuroreceptors

NT

pore Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-Cl-

Cl-Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-Cl-

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Ligand-gated ion channel neuroreceptors

NT

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Ligand-gated ion channel neuroreceptors

NT

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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 others101

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 T-tubules 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 Plasmid2. Lipofectamine complexing3. Add to Dishes4. 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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Proposed conformational shift of A-helix caused by substitution of Proline for G219Prolines 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 adapting the prefered helical geometry

Conserved glycineIn the S6 domain

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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-and-

chain 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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• 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

Na+ Channel Modulation

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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

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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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