Bio2000

Diffusible and electrical signaling factors

Presented by:

Michael Xi Zhu
Department of Neuroscience and
Center for Molecular Neurobiology
The Ohio State University
168 Rightmire Hall
1060 Carmack Road
Columbus, Ohio 43210
614-292-8173
zhu.55@osu.edu
A. Diffusible messengers for signal transduction

Effector
G G

1st messenger
kinases
2nd messenger
 Criteria of second messengers

• 1. When applied intracellularly, it mimics the effect of an

extracellular stimulus.
• 2. It can be synthesized and metabolized in a range of
tissues from many species.
• 3. Antagonism of the action of the messenger blocks the
effects of the extracellular messenger
• 4. Specific intracellular binding sites are present.

• 5. Its level is responsive to physiologically relevant stimuli.

 Diffusible messengers: how many are there?

• Cyclic nucleotides and sugar nucleotides: cAMP, cGMP,

cADP-ribose, NAADP, ADP-ribose
• Phosphoinositides and derivatives: PIP2, IP3,
diacylglycerol, PIP3
• Calcium ion
• Arachidonic acid and metabolites
• Nitric oxide (NO), carbon monoxide (CO), ROS
• Ceramide, Sphingosine, Sphingosine-1-P
• Platelet-activating factor (PAF)
• Anandamide
Cyclic nucleotides and sugar nucleotides: cAMP

Adenylyl Cyclase

NH2
S-S
extracellular

intracellular
α βγ
P G
P
NH2
COOH

COOH
receptor G protein effector
Cyclic nucleotides: cAMP formation, Gs

OH
Adenylyl Cyclase
OH
OH
OH +H N
3
OH
+H N
3 OH
OH

NH2
OH

+H N
3
OH

S-S
extracellular

intracellular αsG
P
P
γ
β NH2
COOH

COOH
receptor G protein effector
Cyclic nucleotides: cAMP formation

OH
Adenylyl Cyclase
OH

+H N
3
OH

NH2 OH
OH

+H N
S-S
3
OH

extracellular OH
OH

H3 N
OH

intracellular P
αsG
P
γ
β NH2
COOH

COOH

receptor G protein effector

Cyclic nucleotides: cAMP formation

OH
Adenylyl Cyclase
OH

+H N
3
OH

OH
NH2
OH

+H N
3 S-S
OH

extracellular OH
OH

H3 N
OH

intracellular αs γ
P
P G β NH2
COOH
PPG
P

COOH
receptor G protein effector
Cyclic nucleotides: cAMP formation

OH
Adenylyl Cyclase
OH

+H N
3
OH

OH
NH2
OH

+H N
3 S-S
OH

extracellular OH
OH

H3 N
OH

intracellular P
PPG
αs γ
β NH2
COOH
P G
P

COOH
receptor G protein effector
Cyclic nucleotides: cAMP formation

OH
Adenylyl Cyclase
OH

+H N
3
OH

OH
NH2
OH

+H N
3 S-S
OH

extracellular OH
OH

H3 N
OH

intracellular
NH2 γ αs
PPG

β
P
COOH
P G
P
NH2 NH2
COOH P P
N N
N N
receptor O O O
N N
N N
O P O P O P O CH2 CH2
O O
O O O O
ATP O cAMP
OH OH P O OH
O
Cyclic nucleotides: cAMP removal

OH
Adenylyl Cyclase
OH

+H N
3
OH

OH
NH2
OH

+H N
3 S-S
OH

extracellular OH
OH

H3 N
OH

intracellular
NH2 γ αs
PPG

β
P
COOH
P
NH2 NH2
COOH P P
N N
N N
receptor O O O
N N
N N
O P O P O P O CH2 CH2
O O
O O O O

ATP O cAMP
OH OH P O OH
O
Cyclic nucleotides: cAMP removal

Adenylyl Cyclase

NH2
S-S
extracellular

intracellular
γ NH2 αsG
P
P

β COOH

P
NH2
COOH
N
N
receptor N N
CH2
O
O
O cAMP
P O OH
O
Cyclic nucleotides: cAMP removal

Adenylyl Cyclase

NH2
S-S
extracellular

intracellular αsG
P
P
γ
β NH2
COOH

COOH
receptor G protein effector

NH2 NH2

5’-AMP N
N N
N
O
cAMP
O P O CH2
N N phosphodiesterase CH2
N N
O O
O O
O cAMP
OH OH P O OH
O
Cyclic nucleotides: inhibition of cAMP formation, Gi

OH
Adenylyl Cyclase
OH

+H N
3
OH

OH
NH2
OH

+H N
3 S-S
OH

extracellular OH
OH

H3 N
OH

intracellular
NH2 γ P P G
αi/o
β
P
COOH
P G
P
NH2 NH2
COOH P P
N N
N N
receptor O O O
N N
N N
O P O P O P O CH2 CH2
O O
O O O O
ATP O cAMP
OH OH P O OH
O
Cyclic nucleotides: cAMP, diversity of adenylyl cyclases

ATP cAMP
NH2 NH2

N N
N N
P P
O O O
N N N N
O P O P O P O CH2 CH2
O O
O O O
O
O
OH OH adenylyl P O OH
cyclase O

Group A Group B Group C

Stimulated by Synergy with “Typical”
Ca2+-calmodulin inhibitory G proteins

Gs Gq, etc. Gs Gi/o Gs

αs Ca2+-calmodulin αs βγ αs

AC I (III, VIII) AC II (IV) AC V (VI)

 Cyclic nucleotides: cAMP targets

Protein kinase A

C C C
R + cAMP R - cAMP R
+ active
R R after short R
C C activation C
period

after long
proteolysis activation
period

R C

R C

remain active
 Cyclic nucleotides: cAMP targets

cAMP-regulated guanine nucleotide exchange factors (cAMP-GEPs)

or exchange protein directly activated by cAMP (Epac)
Cyclic nucleotides: cAMP targets

Olfactory cyclic nucleotide-gated

channels (CNG)

NH2
Odorant molecular
. .. . . ..
S-S
.. . .
extracellular
..
intracellular γ
β NH2
P
P P G
αolf COOH

. .
P G

.
P
COOH
Odorant receptor P P

cAMP NH2
. .
ATP
.
N
N
NH2
N N N
N CH2
O
O O O O
N N
O P O P O P O CH2 O
O
O O O P O OH
O
OH OH
Cyclic nucleotides: cGMP formation
O O

GTP N
NH cGMP N
NH
O O O
N
P P
N NH2 N N NH2
O P O P O P O CH2 CH2
O O
O O O O
O
OH OH guanylyl P O OH
cyclase O

Intracellular Ca2+ CaM Membrane Bound

Guanylyl Cyclase
Ca2+ Stores Ca2+
NO GTP
Soluble
Ca2+
NO
Synthase NO Guanylyl Cyclase
+
Citrulline GTP PDE
cGMP GMP
Arginine
Ion Channels
cGMP-Dependent PK
PDE Activity
 Cyclic nucleotides: cGMP formation

Membrane-bound guanylyl cyclase Soluble guanylyl cyclase

Extracellular Domain

Membrane N-Terminal Segments

Kinase Homology Domain

Central Dimerization Region
Hinge Region
Cyclase Homology Domain
Guanylyl Cyclase Domain

Kuhn M. 2003, Circ Res 93:700-709

GTP
cGMP
Andreopoulos S and Papapetropoulos A, 2000,
Gen Pharmacol 34:147-157
GC-A to GC-G (atrial natriuretic peptide-A, B,
C, guanylin, uroguanylin, and heat-stable α1, α2, α3
enterotoxin) β1, β2, β3

Photoreceptor guanylyl cyclases [ROS-GC1

(retGC1, GC-E); ROS-GC2 (retGC2, GC-F)] Activator: NO
(low Ca2+, GCAPs)
Cyclic nucleotides: cGMP removal

cGMP phosphodiesterase

. .. ... . . ..
Photon

. . . . . .. . .
NH2

S-S
extracellular
CHO

intracellular γ αt
PPG
β P
PDE
P G
P
rhodopsin COOH
O O

5’-GMP N
NH N
NH
O
N N NH2
cGMP N
O P O CH2 CH2 N NH2
O O
O O
O
OH OH P O OH
O
Cyclic nucleotides: cGMP targets

1) cGMP-dependent kinases (PKG)

Two types:
PKG I -- cytosolic
PKG II -- membrane bound

2) cGMP-dependent phosphodiesterases (PDEs)

many isoforms:
cGMP can activate or inhibit PDEs
Can be PDE specific for cGMP, cAMP, or Both

3) cyclic nucleotide-gated channels

 Cyclic nucleotides and sugar nucleotides:
cADP-ribose and NAADP, formation

(nicotinic acid adenine dinucleotide phosphate) Cancela, Annu. Rev. Physiol. 63, 99-117, 2001
 Cyclic nucleotides and sugar nucleotides:
cADP-ribose and NAADP, removal

• cADP ribose: cADP hydrolase

• NAADP: alkaline phosphatase
Cyclic nucleotides and sugar nucleotides:
cADP-ribose and NAADP, targets

• cADP-ribose:
RyR, endoplasmic
reticulum
• NAADP:
NAADP sensitive
Ca2+ stores (NAADP
receptor), lysosome-
like organelles

Lee HC, 2003 Curr Biol 13:R186-188

 Sugar nucleotides: ADP-ribose

ADP-ribosyl-
transferase
Formation: NAD ADP-ribose + nicotinamide

cADP-ribose
hydrolase
cADP-ribose + H2O ADP-ribose

ADPR pyro-
phosphatase
Removal: ADPR AMP + ribose 5’-phosphate

Target: ion channel (TRPM2)

 Phosphoinositides and derivatives

G protein-coupled receptors
OH
OH

+H N
3
OH

OH OH

OH
NH2 OH
NH2 OH

OH +H N +H N
3 3

S-S
OH OH
+H N
3
OH
S-S
extracellular OH
OH
OH
OH

H3 N
H3 N
OH
OH

intracellular
αi
P
PPG
γ αq
PPG γ
β
P
β
P G P G
P P
COOH PLCβ COOH

α2 receptor α1 receptor

Receptor or non-receptor tyrosine kinases

extracellular

intracellular P P P Src
P P
EGFR

PLCγ
Phosphoinositides and Phospholipase C activated signaling
fatty acid chain of outer lipid monolayer of plasma membrane

fatty acid chain of inner

lipid monolayer
of plasma membrane

O O O O O O O O
O O O O O O O O
H2C C
H
CH2 H2C C
H
CH2 H2C C
H
CH2 H2C C
H
CH2 PKC
O O O OH
P P P
1 1 1
4
5
4
5
4
5
P DAG
P P

PI PIP PIP2
ATP ADP ATP ADP Phospholipase C

P P
1
5
5’-Phosphatase 1
5

Ca 2+
4 P 4
P P

I(1,4,5)P3 I(1,4)P2

Ca2+

ER Ca2+ store
 Phosphoinositides and derivatives: PI cycle

cyclic Ins(1:2,4,5)P3 InsP6

Ins(1,3,4,6)P4
cIns(1:2,4)P2
PI(3,4,5)P3 PI(4,5)P2 Ins(1,3,4,5)P4 Ins(1,3,4,5,6)P5
Ins(1,3,4)P3
DAG Ins(1,4,5)P3
cIns(1:2)P

PI(3,4)P2 PA Ins(1,4)P2 Ins(1,3)P2

Ins(3,4,5,6)P4
PI4P Ins(3,4)P2
Ins(1)P
Ins(4)P Ins(1)P
CDP-DG LiCl
Ins(3)P
PI3P myo-inositol
PI

OH
P HO
P
OH P

Ins(1,4,5)P3
Phosphoinositides and derivatives: DAG targets

PIP2 Ca2+
DAG IP3
• Protein kinase C
PA IP2 • RasGRP (Ras guanine-
PIP
nucleotide-releasing protein)
IP
CDP-DG LiCl • chimaerins
myo-inositol • ion channels (e.g. TRPC)
PI
Phosphoinositides and derivatives: PIP2 targets

PIP2 Ca2+
DAG IP3

IP2 K+ channels: Kv, Kir

PA PIP Ca2+ channels: VGCCs and TRPs
IP CNG
CDP-DG LiCl IP3 receptors?
myo-inositol
PI
Phosphoinositides and derivatives: IP3 targets

IP3 receptors
PIP2 Ca2+ Other IP3 binding proteins
DAG IP3

PA IP2
PIP
IP
CDP-DG LiCl
myo-inositol
PI

OH
P HO
P
OH P

Ins(1,4,5)P3
Phosphoinositides and derivatives: may be more complicated

63 inositol phosphate esters

Shears S. B. 1998, Biochim Biophys Acta 1436:49-67

Phosphoinositides and derivatives: PIP3 targets

PI(3,4,5)P3 PI(4,5)P2 Ca2+

DAG Ins(1,4,5)P3

PI(3,4)P2 PA Ins(1,4)P2
PI4P
Ins(4)P
CDP-DG LiCl

PI3P myo-inositol
PI
• Serine/threonine kinase
Akt (protein kinase B, PDK1, RAC-PK)
p70 ribosomal S6 kinase (p70S6K)
PKC
• Ion channels?

PDK1: PI(3,4)P2/PI(3,4,5)P3 dependent kinase 1

RAC-PK: Related to PKA and PKC protein kinases
 Ca2+ signaling: sources
Plasma membrane
Ca2+ Ca2+ Ca2+ permeable channels
Ca2+
voltage-gated Ca2+ channels
Ca2+
Ca2+
ionotropic receptor/cation channels
cyclic nucleotide-gated channels
extracellular space Ca2+ Ca2+
Ca2+ store-operated channels Ca
2+

[Ca2+]o = 10-3 M other non-selective cation channels

Ca2+
Ca2+
Ca2+v

+ + + + + + + + + + + + + + + + + + + + + +

- - - - - - - - - - - - - - - - - - - - - -
cytosol [Ca2+]i = 10-7 M Ca2+

Intracellular Ca2+ channels

Ca2+ IP3 receptors
Ca
2+
Ca2+ ryanodine receptors
Ca2+
NAADP receptor?
Ca2+
Ca2+ PTP? (MT)
Ca2+
endoplasmic reticulum
Ca2+
[Ca2+]ER = 10-4 ~10-3 M
 Ca2+ signaling: removal
Ca2+
Plasma membrane extrusion
Ca2+
Ca2+
Ca2+-ATPases (PMCA1-4)
Ca2+
Ca2+ Na+/Ca2+ exchanger (NCX1-3)
extracellular space Ca2+ Ca2+
Ca2+
Ca2+

[Ca2+]o = 10-3 M Ca2+

Ca2+
Ca2+v

+ + + + + + + + + + + + + + + + + + + + + +

- - - - - - - - - - - - - - - - - - - - - -
cytosol [Ca2+]i = 10-7 M Ca2+

Cytosolic Ca2+-binding proteins:

Calmodulin, Neuronal Ca2+ sensors
Synaptotagmin, Phospholipase C
Ca2+ Protein kinase C, Calbindin, Calretinin
Calreticulin (CRT),Ca
2+

Parvalbumin
Calsequestrin (CSQ) Ca 2+

Calnexin, Junctate, Bip, Grp94, Intracellular Ca2+ reuptake

Reticulocalbin, ERC55, Cab55
Calumenin,CaCrocalbin Ca
2+ 2+ Ca2+-ATPases (SERCA1-3)
endoplasmic reticulum Ca2+
Ca2+
mitochondrial uniporter
Ca2+
[Ca2+]ER = 10-4 ~10-3 M
 “On” and “Off” mechanisms of intracellular Ca2+ signal

Stimulus
Ca2+ 1 mMPlasma membrane Ca2+ channels
Plasma
membrane
R R RTK R ∆V
G
Cytoplasm ? ? PtdIns(4,5)P2 PLCβ Ca2+ sensitive processes
NAD TnC Contraction
NADP PLCγ
CAM MLCK
Sphingosine
Ins(1,4,5)P3 CAMKs Proliferation
Calcineurin Fertilization
ADP ribosyl cADPR Transcription Learning and
cyclase NAADP
factors memory

S1P Crosstalk
Sphingosine Adenylyl cyclase with other
kinase Cyclic AMP PDE signaling
NOS pathways
PKC
PMCA SERCA PYK2
Ca2+
2+ IP3 3-kinase
Ca2+ - - Ca2+ Membrane
~100 µM ~1,000
~100 nMnM Ion channels excitability
IP3R, RyR,
Na+ SCaMPER Secretion
ER/SR Synaptotagmin
NAADPR Metabolism
Ca2+ Buffers/chaperones Phosphorylase
kinase Vesicle
Na /Ca
+ 2+ trafficking
Exchanger Ca2+ buffers Annexin family Cell
proliferation
Mitochondrion
S100 family Cell
proliferation,
Na+/Ca2+ cancer and
Exchanger Na
+
metastasis
Ca2+ Mitochondrial ATP synthesis
enzymes Steroid synthesis

PTP Cytochrome c Caspases Apoptosis

Berridge et al., 2000, Nature Rev. Mol. Cell. Biol. 1, 11–21.

Ca2+ Signaling: targets

Ca2+ binding proteins: Ca2+ binding motif:

Channels and enzymes: EF hand
Annexins C2 domain
Ca2+ channel
K+ channel
PKC
Calpain

Calmodulin
NMDA receptor, olfactory CNG channel, Ca2+ channel, K+
channel, Na+ channel, TRP channel
PDE
Adenylyl cyclase
Plasma membrane Ca2+-ATPase
NO synthase
Calcineurin
CaM kinase and other kinases
EF hand

Chicken Troponin C site 4

 Calmodulin:

substrate

EF-hand

CaMKII peptide
Ca -binding
2+

domain

Calmodulin

Mechanism of action
CaM

A
A B A B

CaM
B
Modes of intracellular [Ca2+] changes: oscillation

Frequency-dependent cellular function

Sustained elevation Low-frequency oscillation High-frequency oscillation

stimulus stimulus stimulus
[Ca2+]i

[Ca2+]i

[Ca2+]i
Time Time Time
Modes of intracellular [Ca2+] changes: Ca2+ sparks

F/F0

Stephen Baylor, M.D., University of Pennsylvania

F/F0

F/F0

500 ms
Mark T. Nelson, Ph.D, University of Vermont
Modes of intracellular [Ca2+] changes: Ca2+ waves

Ca2+ wave triggered by a Ca2+ spark in a single myocyte

Subramanian et al., 2001, Biophys. J. 80, 1-11

Modes of intracellular [Ca2+] changes: Ca2+ waves

Ca2+ waves in differentiated PC12 cell stimulated with bradykinin propagates

from the tip of neurite to soma. [Ca2+]e = 0

Lorenzon et al., 1995, J. Cell Biol. 129, 797-804

Spiral Ca2+ wave in a Xenopus oocyte

injected with a nonhydrolyzable analog
of IP3 (Confocal Fluo3 image)

Lechleiter and Clapham, 1992, Cell, 69, 283-294.

Relationships between different Ca2+ release channels

NO/cGMP

Cancela, Annu. Rev. Physiol. 63, 99-117, 2001

 Arachidonic acid and metabolites: formation
DAG PA
O O
O CH2 O C R1 O CH2 O C R1
DAG kinase
R2 C O CH R2 C O CH O
CH2OH PA phosphohydrolase CH2 O P OH
O-
DAG lipase H2O
H2O X
O H2O +
CH2CH2 N(CH3)3
O XOH PC:
O
X O P O PLC
-
O CH2 O C R1 PLD PE: CH2CH2
+
NH3
R2 C O-

O- R2 C O CH O +
NH 3
O CH2 O P OX PS: CH2CH
CH2 O C R1 H2O H2O
O -
COO-
HO CH
CH2OH PLA1 PI: 1
PLA2 4
5

O
O CH2OH O O CH2 O C R1
R2 C O CH O O- C R1 R2 C O- HO CH O
CH2 O P OX CH2 O P OX
O- H2O H2O O-

PLL2 PLL1
CH2OH
HO CH O
O O
CH2 O P OX
R2 C O- O- C R1
O -
Arachidonic acid and metabolites: formation and removal

hydroperoxyeicosatetraenoic acids dihydroxyeicosatetraenoic

acid

hydroxyeicosatetraenoic acids

epoxyeicosatrienoic acid

thromboxane A 2
prostaglandins leukotrienes
Arachidonic acid and metabolites: targets

AA: ion channels, Kv4 (A current), Kir2.3, TRP, ARC

Cav, ClC-2, KCa, TREK and TRAAK

EET: ion channels, SOC, TRPV4

HPETE: TRPV1
Nitric oxide (NO): formation

NO Synthases:
NOS1: neuronal or nNOS
NOS2: inducible or iNOS
NOS3: endothelial or eNOS
Nitric oxide (NO): removal and targets
3 different forms of NO:
.
NO (NO free radical)
NO- (Nitrosyl anion)
NO+ (Nitrosonium)
Second messenger characteristics:
Quickly made and destroyed
Lipophilic
Gaseous
Highly reaction free radical
• Reduction reactions – occur in the presence of O2
-- Lead to end products NO2-, NO3-
-- Rapid interconversion of nitrogen oxides
• Reactions with transition metals
-- Porphyrin rings (soluble guanylyl cyclase)
-- Heme groups
• Reactions with superoxides
-- formation of oxidative stress molecules
(e.g. peroxynitrite: OONO-)
• Reactions with amines and thiols
-- protein modification for long term signaling
 Nitric oxide (NO): signaling

• Before 1985, NO was considered primarily as an air pollutant

• NO is an established messenger in physiological processes, such as:

Vascular Tone
Platelet aggregation
Host defense
Inflammation
Neurotransmission
Cell migration
Learning and memory
Penile erection
Hormone release
Cell differentiation
Apoptosis
Gastric emptying

• Nobel Prize in Medicine, 1998

B. Electrical signaling
Origin of resting membrane potentials
uneven ion concentrations inside and outside the plasma membrane and
selective permeability to different ions.

E=0
- E = Ek +

- +
- +
- +
K+ K+ K+ -
-
+ K+
+
- +
A- A- A- -
-
+ A-
+
- +

Time zero At equilibrium

RT [S]2
Nernst equation: ES = E1- E2 = ln
zSF [S]1
Resting membrane potentials
Nernst equations for biological ions: Anions: Cl- and proteins
Cations: K+ diffusion potential
RT [K]o Na +
diffusion potential
Ek = ln
F [K]i Ca2+ diffusion potential
RT [Na]o Na+/K+-ATPase
ENa = ln
F [Na]i
RT [Ca]o
ECa = ln
2F [Ca]i
RT [Cl]o
ECl = ln
-F [Cl]i
-60 to -75 mV
extracellular ENa = +56 EK = -102 ECl = -76 ECl = +125 NSCC
Na+ (150) K+ (3) Cl- (120) Ca2+ (1.2)

intracellular Na+ (18) K+ (135) Cl- (7) Ca2+ (0.1 µM)

Na+,K+-ATPase
 Maintaining the resting membrane potential

Negative to EK Positive to EK
gK
K+ moves out of cell
Intracellular Extracellular

- K + EK
Reversal potential
(No net movement of K+)
- K
+
K
K
- K + K+ moves into cell
gK
- K
K
+
Concentration gradient Time
- K
voltage gradient +
K
- K K + Normal
K Increased gK
- K + current injection
K
- +
K
- K
+ Voltage response K+ moves out of cell

EK
-102 mV -102
gK K+ moves into cell

Time
 Maintaining the resting membrane potential

The Goldman-Hodgkin-Katz Equation:

The steady state membrane potential for a given set of ionic concentrations inside
and outside the cell and the relative permeability of the membrane to each ion

RT pK[K+]o + pNa[Na+]o + pCl[Cl-]i

Vm = ln
F pK[K+]i + pNa[Na+]i + pCl[Cl-]o

-60 to -75 mV
extracellular ENa = +56 EK = -102 ECl = -76 ECl = +125 NSCC
Na+ (150) K+ (3) Cl- (120) Ca2+ (1.2)

intracellular Na+ (18) K+ (135) Cl- (7) Ca2+ (0.1 µM)

Na+,K+-ATPase
Changes in membrane potential due to ion movement

Depolarization:

Initiators: Na+ channels

nonselective cation channels (NSCC)
Na+,K+-ATPase

Terminators: K+ channels
Cl- channels

-60 to -75 mV
extracellular ENa = +56 EK = -102 ECl = -76 ECl = +125 NSCC
Na+ (150) K+ (3) Cl- (120) Ca2+ (1.2)

intracellular Na+ (18) K+ (135) Cl- (7) Ca2+ (0.1 µM)

Na+,K+-ATPase
 Types of electrical signals
• Graded potentials: Variable-strength signals that lose strength as they travel
through the cell.
a. Can be depolarizations (Na+ channel) or hyperpolarizations (K+ or Cl- channel)
b. Begins on the cell membrane at the point where ions enter from the extracellular
fluid (local current or electrotonic current)
c. The strength or amplitude is directly proportional to and is determined by the
number of charges that enter the cell, which in turn is determined by the number of
receptors which are opened. (concentration of the neurotransmitters and density of
the receptors)
d. The size of the graded potential decreases as it spreads out from its point of origin
e. Graded potentials travel through the neurons until they reach the trigger zone, the
point where an action potential is generated. Depending on the strength of the
graded potential, it either triggers an action potential or dies out (threshold
potential).
f. Can be summed: spatial summation and temporal summation

• Action potentials: Signals that travel for long distances through the neuron
without losing strength.
a. Rapid electrical signals that pass along the axon to the axon terminal.
b. Identical to each other and do not diminish in strength when traveling through the
cell
c. The strength of the graded potential that initiates an action potential has no
influence on the action potential as long as it is above threshold.
d. All-or-none
 Comparison of graded potential and action potential
Feature
Type of signal
Where it occurs
Types of gated ion channels
Ions involved
Type of signal
Strength of signal
What initiates the signal
Unique characteristics
Graded Potential
Input signal
Usually dendrites and cell body.
Mechanically or chemically gated
channels
Usually Na+, K+, and Cl-
Depolarizing (Na+ ) or hyperpolarizing
(K+, Cl- )
Depends on initial stimulus; can be
summed
Entry of ions through chemically or
mechanically gated ion channels
No minimum level required to initiate
a graded potential
Two signals coming close together in
time will sum
Action Potential
Conduction signal
Axon hillock, initial segment and entire
length of axon
Voltage-gate channels
Na+ and K+
Depolarizing
Is always the same as long as graded
potential is above threshold; cannot be
summed
Above-threshold graded potential arrives
at the integration zone
Threshold stimulus required to initiate
action potential
Refractory period: two signals too close
together in time cannot sum
Initial stimulus strength is indicated by
frequency of a series of action potentials
Spread of steady state signal in axons
Model electric circuit for
a nerve segment At steady state

ri ri ri ri
. . . .

.
rm
rm
cm Er

rm . d2V
V=
ri dx2
Characteristic length λ = x when V/V0 is 1/e = 0.37
V = V0e-x/λ
The larger the λ, the further the potential spreads.
rm Rm .d
λ= = The larger the diameter, the further the potential spreads.
ri Ri 4
Ri = specific internal resistance (Ω cm)
Rm = specific membrane resistance (Ω cm2)
 Spread of transient signals

dVm Vm
C + = Ipulse
dt R
dVm V

8
V (1/e)
RC dt + Vm = R.Ipulse

8
V (1/e)

8
τm
Injected current Im

RC = τ current thru ionic leak, Ii

Initial response of the membrane to the

injected current pulse, I is: current thru capacitance, Ic

Vm(T) = IpulseR(1-e-T) T = t/τ

When the pulse terminates, the decay of
the initial potential (V0) to rest is: 0 1 2 3 4 5 t/τm

Vm(T) = V0e-T V : steady state voltage

8
τm: membrane time constant

τ is time constant, i.e. the time required for the voltage

change across the membrane to reach 1/e = 0.37 of its
final value.
Action potentials
Spread of action potentials
Action potentials in mammalian brain
Cortical pyramidal cell
Cerebellar Purkinje cell
Spread of electrotonic potential in an entire neuron
 Spread of electrotonic potentials from cell to cell
• Electric coupling: gap junction

-
- - -
- -
- -

-
- - -
- -
• Chemical coupling: synapse
Presynaptic Postsynaptic
terminal terminal 5 mV
20 ms

ICa
α1B
NMDAR

Ca2+ AMPAR
Ca2+
DAG
PLCβ4

Gq/11
IP3
Ca2+
mGluR1
 Voltage-gated ion channels: currents
Inward currents Outward currents

-10 -10 Voltage (mV) -10

-100 Voltage (mV) -100 -100 Voltage (mV)

Na+ Na+/K+ K+
INa,t Ih IK

IA
INa,p
IM
Ca2+
ICa,L
IC
ICa,N

ICa,T
Voltage-gated ion channels: structure

Perez-Reyes, Cell Mol Life Sci 56, 660-669, 1999

Voltage-gated ion channels: structure of Ca2+ channels

Skeletal muscle L-type Cardiac muscle L-type

α2
α1s δ
γ

β
β

Bers and Perez-Reyes, Cardiovasc. Res. 42, 339-360, 1999

Voltage-gated ion channels: family of Ca2+ channels

10 types of α1 subunits

CaV1.1 (α1S)
*C48a7 CaV1.2 (α1C) L
CaV1.3 (α1D)
CaV1.4 (α1F)
P/Q
*unc2 CaV2.1 (α1A)
N
CaV2.2 (α1B) R
CaV2.3 (α1E)
*C54d2 CaV3.1 (α1G) T
CaV3.2 (α1H)
Ca 3.3 (α1I)
20 40 60 80 100 V
Matching percentage using CLUSTAL

* C. elegans genes
Modified from Ertel et al, Neuron 25:533-535, 2000.
 Ligand-gated ion channels (ionotropic receptors)

Bind to neurotransmitters
Receptor channels
Mediate fast synaptic transmission

Postsynaptic
. .. . .
Presynaptic
ions terminal terminal 5 mV
20 ms

ICa
α1B
NMDAR

Ca2+ AMPAR
Ca2+
.. DAG

PLCβ4
ligand

. Gq/11
IP3

. . Ca2+
mGluR1
ionotropic glutamate receptors

10% GluR1
GluR2 AMPA:
GluR3
α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid
GluR4
GluR5
GluR6
GluR7
KA1
kainate
KA2
NR1
NR2A
NR2B NMDA: N-methyl-D-aspartate
NR2C
NR2D
Coincidence detector because of voltage dependent
Mg2+ block
NH2

TM4

NR1 TM3 NR1 TM1

NR2
NH2 NR2 TM1 TM3
extracellular NR1 TM1 TM4
TM4 NR2 Mg 2+
NR2 TM4
TM3
TM2 TM3 TM1
TM1 TM4
TM2 TM3 TM1 NR1 TM3
intracellular
TM4
COOH

COOH
 Cys-loop superfamily
Muscle-type Neuronal type

Cation channels
Nicotinic acetylcholine receptor
I, epithelial α9;
II, neuronal α7,8;
subfamilies

III, neuronal α2–6 and β2–4

III-1: α2,3,4,6; homo-oligomeric α7
NH2
III-2, β2,4 COOH
III-3, α5, β3;
IV, muscle α1, β1, γ, δ, and ε extracellular

IV-1, α1 TM1 TM2 TM3 TM4

IV-2, γ, δ, ε intracellular

IV-3, β1. hetero-oligomeric α4β2

Corringer et al., Annu. Rev. Pharmacol. Toxicol. 40:431-458, 2000
5-HT3 serotonin receptor
5-HT3A, 5-HT3B
α γ TM4
TM3 TM4
β α TM1
γ
Anion channels α
TM1
TM2 TM2 TM3
δ NH2
GABAA receptor TM2
TM1
TM4
TM3
TM1
COOH
TM3
TM4 β TM2
Glycine receptor TM1 TM2
TM2 α TM4

TM3 δ TM1
TM3

TM4
 P2X receptors

a P2X2
b
P2X3
Cysteine rich
extracellular P2X5
loop P2X6
P2X1
P2X4
Plasma membrane
P2X7

NH2 Each channel may

contain three to six
subunits
• Activated by ATP
• Cation nonselective
C tail length varies
• ~6.5% of current is carried by Ca2+

Khakh et al., Pharmacol. Rev. 53, 107-118. 2001

Other channels

• Mechanosensitive channels (e.g. TRPs)

• Temperature sensitive channels (e.g. TRPs)
• Second messenger-gated channels (e.g. CNGs, ARC)
• Proton-gated channels (ASICs, TRPVs)
• Inwardly rectifying K+ channels
• Ca2+-activated channels (KCa, ClCa, NSCCCa)
• Store-operated channels (TRP)
• CFTR
TRP (Transient Receptor Potential) superfamily
TRPC2 (Trp2)
TRPC3 (Trp3)
TRPC7 (Trp7) TRP-Canonical
TRPC6 (Trp6) (Store-operated,
TRPC4 (Trp4) Receptor-operated)
TRPC5 (Trp5)
TRPC1 (Trp1)
TRPV1 (VR1, capsaicin receptor)
TRPV2 (VRL1, GRC, high-heat receptor)
TRPV4 (OTRPC4, Trp12, VR-OAC)
TRPV3 (Intermediate heat receptor)
TRPV6 (CaT1, ECaC2, CaT-L, SOC?)
TRPV5 (ECaC1, CaT2)
TRPM2 (TRPC7, LTRPC2, ADP-ribose activated)
TRPM8 (Trp-p8, CMR1, cold receptor)
TRPM5 (Mtr1, LTRPC5, Ca2+-activated, role in taste)
TRPM4 (FLJ20041, LTRPC4, Ca2+-activated)
TRPM3 (KIAA1616, LTRPC3)
TRPM1 (Melastatin, MLSN1, LTRPC1)
TRPM6 (ChaK2)
TRPM7 (TRP-PLIK, ChaK1, LTRPC7,
Mg2+/ATP-inactivated )
TRPV1 (vanilloid receptor 1 or capsaicin receptor)
A polymodal detector of noxious pain stimuli and the mediator of inflammatory pain

Noxious stimuli Inflammatory mediators

Prostaglandins
resiniferatoxin

capsaicin
Bradykinin

anandamide

Histamine

High temperature Low pH ATP

Diffusible and electrical signaling factors for TRPV1 function

Heat
Vanilloids
Neurotransmitters H+ Neurotransmitters
Inflammatory factors Inflammatory factors
5 mV
20 ms Peptides Na+, Ca2+ Peptides
NGF ATP

VGIC GPCR GPCR GPCR TRPV1 GPCR

AC
P P
P P PLC G PLA2 G
q/i
PLD G ATP P Gsi
q/11 q/i

PIP2
cAMP
Anandamide
LM
prostaglandins
IP3
AA
DAG PKC PKA
Ca2+
ER