Transcript use-dependent blocker

Reminder: Henry Lester’s “office” hours outside the Red Door
Mon, 1:15-2 PM, Fri 1:15-2 PM
Bi 150 Lecture 7
Monday, October 12, 2015
Postsynaptic acetylcholine receptors;
Synaptic transmission is a δ-function in time;
Channel blockers
Chapters 9, 12
1
Timing of synaptic events
“Synaptic delay”, between the
peak of the action potential and
the start of transmitter release, is ~
0.5 ms.
Delay between the peak of the Ca2+
current and the beginning of the
EPSP is ~ 0.2 ms (more at lower
temperature).
Most of the “synaptic delay” is
caused in opening of Ca2+
channels during the action
potential.
mV
The size and timing of the
EPSP’s can be modulated by
prolonging the action potential.
Figure 12-1
2
Endplate Potential is not Regenerative
(contrast to Action Potential)
The current source for the
epp is restricted to the
endplate, so the size of the
potential decays with
distance from the endplate
(space constant ~ 1 mm).
Figure 9-5
3
electrical
transmission in
axons:
electric field
open
closed
Past lectures:
V-gated Na+ channels
V-gated K+ channels
V-gated Ca2+ channels
chemical
transmission at
synapses:
[neurotransmitter]
closed
open
Today: ACh-gated excitatory cation (Na+ / K+ / Ca2+) channels,
Future: GABA and glycine-gated inhibitory anion (Cl- channels)
Future: Glutamate-gated excitatory (Na+ / K+ / Ca2+) channels
4
From the previous lecture
Many basic principles of
chemical transmission
were discovered at the
neuromuscular junction
(nerve-muscle synapse,
endplate);
acetylcholine is the
transmitter.
Figure 9-1
5
From previous lectures
Fine structure of the
nerve-muscle synapse
0.3 µm
Incl. acetylcholinesterase
ACh receptors
Figure 9-1
6
The family of nicotinic ACh Receptors
The mammalian genome contains 10  subunits (1 through 10) and
3  subunits (1 through 4), γ, δ, and ε subunits.
Nicotinic receptors always contain at least two  subunits. Muscle: (α1)2β1εδ
Major brain ACh receptors: α4β2 (stoichiometry uncertain); (α7)5
Mouse Midbrain dopaminergic cells (coronal section, tyrosine hydroxylase stain)
Substantia nigra pars compacta (controls motion); lost in Parkinson’s Disease
A10 Ventral tegmental area (VTA, controls reward)
Who introduced
nicotine to
European
culture?
Hint: which
Holiday is
today?
Substantia nigra pars reticulata (GABAergic)
These neurons express many types of nAChR subunits and combinations
7
Nicotinic Acetylcholine Receptor (Unwin, 2005)
~ 2200
amino acids
in 5 chains
(“subunits”),
Binding
region
MW
~ 2.5 x 106
Membrane
region
Colored by
secondary
structure
Colored by
subunit
(chain)
Cytosolic
region
8
The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004)
Showing the cation-p interaction
Y198
C2
W149
B
Y93
A
Y190
C1
non-W55
D
(Muscle Nicotinic numbering)
9
How do Nicotinic Receptors Transduce Agonist Binding into Channel Gating?
Swivel?
acetylcholine
or
nicotine
acetylcholine
or
nicotine
Miyazawa
& Unwin,
Nature
2003
CLOSED
Twist?
Corringer
et al.,
J Physiol
2010
OPEN
In any case, the permeant ions experience a water-like environment
CLOSED
Hydrophobic Leu (Gate)
OPEN
Polar (Ser or Thr) –OH side chains
(charge selectivity occurs in > 2 regions)
11
The Reversal Potential (Erev = EEPSP) for the synaptic potential (or current):
not a True Equilibrium / Nernst Potential
Membrane
potential
+80
ENa
+60
+40
Most excitatory ligandgated channels allow flux
of Na+ and K+
(sometimes Ca2+), and
Erev ~ -5 mV.
Positive to Erev, agonist
pushes voltage more
negative.
outside
GEPSP
EEPSP
(~ -5 mV)
GK
EK
(-90 mV)
cytosol = inside
+20
-5
-20
At Erev , the agonist has
little effect on
membrane potential.
-50
Resting -80
potential
EK
-100
Negative to Erev, agonist
pushes voltage more
positive.
resting
potential:
K+ channels
open
Excitatory
postsynaptic
responses:
Na+ / K+
channels
open too
EKGK + EEPSPGEPSP
DV =
GK + GEPSP
Desensitization Occurs
at
Many Ligand-Gated Channels
Free Energy
unbound
voltage-clamp trace from
Xenopus oocyte expressing
α4β2
nicotinic receptors
agonist
Bound states with
increasing affinity
“closed/
resting”
“activated”
Highest
affinity
“desensitized”
Reaction
Coordinate
106
channels
nicotine
20 sec
Time course of Postsynaptic Activation:
Back to Feynman’s Idea
A
sensitive electronic ammeter
a nicotinic acetylcholine receptor
exposed to acetylcholine
5 pA = 104 ions/ms
20 ms
dynamic range:
5 ms to 5 min
1 part in 108
14
How ”tight” is the gigaohm seal?
1. Electrically tight
See next slide
1 mm
Alberts 11-31
© Garland
15
How ”tight” is the gigaohm seal?
1. Electrically tight
pipette wall
R = rl/A
R ~ 109 W;
r = resistivity = 22 W-cm;
l = length = 10 mm;
A = area = 10 mm x t (thickness);
Therefore
t ~ 2 x 10-11 m, or less than 1 Å!
t
membrane
16
How ”tight” is the gigaohm seal?
acetylcholine in the pipette
opens channels in the pipette
2. Chemically tight
acetylcholine
outside the
pipette opens
channels
outside the
pipette
The seal compartmentalizes
molecules.
Molecules outside the pipette do not
mix with molecules inside the pipette
17
Neither Kandel nor Alberts describe Sigworth’s beautiful circuits
sensitive electronic ammeter
A
Little Alberts 12-22D
18
© Garland
from Lecture 2
Max Delbruck
Carver Mead
Richard Feynman
“If you want to measure
small, noisy signals, I have
a Senior who can help”
H. A. L
19
Fred Sigworth ‘74 and Apostol’s Clock
Fred Sigworth’s Web page at Yale
http://bbs.yale.edu/people/fred_sigworth.profile
Ma 1A:
http://www.amazon.com/Calculus-Vol-One-VariableIntroductionAlgebra/dp/0471000051/ref=sr_1_1?s=books&ie=UTF8&qid
=1381358906&sr=1-1&keywords=apostol
20
Statistical analysis of single-molecule events
channel opens
now we synchronize artificially on
the opening event
n =1
0
At a synapse, the pulse of transmitter
is ~ a d-function,
and all postsynaptic channels open nearly
synchronously
Like Figure 9-10
21
Statistical analysis of single-molecule events
n =1
0
Counts/bin
Counts/bin
counts/bin
open time histogram
time constant =
Time
time
1
k 21
closed time
time const
Time
time
22
Molecular lifetimes
from Chem 1b Lecture Series #5
(Heath)
23
Synaptic integration 1A.
Molecular lifetimes
(more in later lectures)
Concentration of
acetylcholine at
nerve-muscle synapse
(because of
acetylcholinesterase,
turnover time
~ 100 μs)
high
State 1
closed
State 2
k21
all molecules
begin here at
t= 0
open
0
units: s-1
Number of open
channels
ms
24
Lidocaine, an example of a drug that blocks voltage-gated or ligand-gated channels
Alkyl substituents:
may adjust
charge density of amine.
Affects membrane
permeation.
Also affects binding to the
receptor.
Charged amine:
may bind to
charged groups or
π electrons on the protein
Amide: hydrolyzed to
terminate drug action
Alky groups affect both
membrane permeation
and receptor binding
Aromatic: may bind to
nonpolar groups on
the receptor protein.
Single-molecule recordings with a lidocaine analog
5 pA
Acetylcholine only
20 ms
Acetylcholine + blocking drug
(QX-222, analog of lidocaine)
26
Drug interactions at the nicotinic acetylcholine receptor
Some drugs compete with acetylcholine
Some drugs bind on the axis
~ 100 Å
(10 nm)
27
Model or
scheme
normal function
State 1
State 2
k21
open
closed
units: M-1s-1
units: s-1
simple block
k21
closed
Functioning channel (1, 2)
all molecules
begin here at
t= 0
open
k23 = k+[Drug]
drug
blocked
Drug-blocked channel (3)
28
current
time constant
= 1/k21
none
time constant
= 1/(k21+ k23)
Functioning channel
Drug-blocked channel
29
Stylized single-channel records for faster- and slow-binding blocking drugs
Open (2)
Closed (1)
blocked (3)
binds
unbinds
30
Lidocaine blocks Na+ Channels from inside the cell
inside
Functioning
channel
“Trapped” or
“Use-Dependent”
Blocker
lidocaine-H+
lidocaine-H+
lidocaine
31
Procainamide, a use-dependent blocker
stimuli
Action potentials fail
impulses
(voltage)
channel
population
(currents)
threshold
pronounced block
at brief intervals
little block
at long intervals
32
inside
Functioning channel
“Trapped” or
“Use-Dependent”
Blocker
33
Na+ channel blockers in medicine
Local anesthetics
Dental surgery (lidocaine)
Sunburn medications
Antiarrhythmics (heart)
“use-dependent blocker”
example: (procainamide)
Antiepileptics / anticonvulsants (brain)
“use-dependent blocker”
(phenytoin = Dilantin® )
34
Superfamilies of ligand-gated ion channels that are synaptic receptors
A. ACh, Serotonin 5-HT3, GABA,
(invert. GluCl, dopamine, tyrosine)
receptor-channels
Most
^
Figure 10-7
35
Reminder: Henry Lester’s “office” hours outside the Red Door
Mon, 1:15-2 PM, Fri 1:15-2 PM
End of Lecture 7
Interested in learning more about how drugs open and block ion channels?
That’s part of neuropharmacology
(Bi 155, Winter 2017)
36