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Bi / CNS 150 Lecture 4

Monday, October 5, 2015

Voltage-gated channels (no action potentials today)

Henry Lester

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http://www.cns.caltech.edu/bi150/

The Bi / NB / CNS 150 2015Home Page

Please note:

Henry Lester’s office hours

Read the book

If you drop the course,

or if you register late,

please email Jaron

(in addition to the Registrar’s cards).

Ion Channels in the News5 October 2015:

“for therapies that have revolutionized the treatment of some of the most devastating parasitic diseases”

Discovered the avermectins (including ivermectin)

Ivermectin irreversibly activates a Cl channelFound among many invertebrates

Next: PDB file 3RIF

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Todays news exemplifies how contributions to neuroscience come from many fields. . . In this cases, parasitology

When ivermectin binds to the invertebrate glutamate-activated Cl- channel “GluCl”, the cell becomes “clamped” at the Cl- Nernst potential (~-70 mV).This prevents the cell from firing action potentials.The parasite cannot feed, and dies.

Vertebrate neurons, engineered to express an optimized GluCl,

enabling the experimenter to “silence” the neuron by applying ivermectin

Transfection:Empty DNAAn older GluCl constructOur optimized “silencing” construct

No IVM

20 nM

Today’s news also exemplifies how electricity is the language of neurons . . .

and of many other excitable cells.

Frazier, Cohen, and Lester J Biol Chem 2013

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In the “selectivity filter” of most K+ channels,

K+ ions lose their waters of hydration and are co-ordinated by backbone carbonyl groups

H2O K+ ion

carbonyl

From Lecture 1

Ion selectivity filter

Gate(Like Kandel Figure 5-15)

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[neurotransmitter]

openclosed

chemical transmission atsynapses:

electric field

openclosed

electrical transmission inaxons:

actually, DE

Major Roles for Ion Channels

Future lectures:

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The electric field across a biological membrane, compared with other electric fields in the modern world

1. A “high-voltage” transmission line1 megavolt = 106 V.The ceramic insulators have a length of ~ 1 m.The field is ~ 106 V/m.

2. A biological membraneThe “resting potential” ~ the Nernst potential for K+, -60 mV.The membrane thickness is ~ 3 nm = 30 Å.The field is (6 x 10-2 V) / (3 x 10-9 m) = 2 x 107 V/m !!!

Dielectric breakdown fields (V/m)

Ceramic 8 x 107

Silicone Rubber 3 x 107

Polyvinyl chloride 7 x 106

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open channel = conductor

Na+ channel

=

From Lecture 1

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1973

Max Delbruck

Richard Feynman

H. A. L

Carver Mead

http://en.wikipedia.org/wiki/Carver_Meadhttp://www.nytimes.com/2015/09/27/technology/smaller-faster-cheaper-over-the-future-of-computer-chips.html

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Intracellular recording with sharp glass electrodes

V

t = RC = 10 ms; too large!

C = 1 mF/cm2

E

R = 104 W-cm2

intracellular

extracellular

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A better way: record the current from channels directly?

Feynman’s idea

A

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5 pA = 104 ions/ms

20 ms

A single voltage-gated Na+ channel

-80 mV

-20 mVA

Dynamic range

10 ms to 20 min : 108

2 pA to 100 nA

50,000 chans/cell

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http://www.nobel.se/medicine/laureates/1991/press.html

Press release for 1991 Nobel Prize in Physiology or Medicine:

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Simulation of Shaker gating

http://nerve.bsd.uchicago.edu/model/rotmodel.html

Francisco Bezanilla's simulation program at the Univ. of Chicago.

“Shaker”, a Drosophila mutant first studied in (the late) Seymour Benzer’s lab

by graduate students Lily & Yuh-Nung Jan (now at UCSF);

Gene isolated simultaneously by L & Y-N Jan lab

& by Mark Tanouye (Benzer postdoc, then Caltech prof, now at UC Berkeley).

“Shaker”, a well-studied voltage-gated K+ channel

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Today we emphasize H & H’s description of channel gating

(although they never mentioned channels, or measured a single channel)

Channel opening and closing rate constants are functions of voltage--not of time:

The conformational changes are “Markov processes”.

The rate constants depend instantaneously on the voltage--not on the

history of the voltage.

These same rate constants govern both the macroscopic (summed) behavior and

the single-molecule behavior.

The Hodgkin-Huxley formulation of a neuron membrane

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This channel is actually Shaker with inactivation removed (Shaker-IR).

Based on biochemistry, electrophys, site-directed mutagenesis, X-ray crystallography,

fluorescence.

Two of 4 subunits. Outside is always above (show membrane). Green arrows = K+.

C1 and C2 are closed states, A is “active” = open.

6 helices (S1-S6) + P region, total / subunit.

Structure corresponds roughly to slide 7,

The two green helices (S5, S6 + P) correspond to the entire Xtal structure on slide 4.

First use manual opening. Channel opens when all 4 subunits are “A”.

Note the charges in S4 (5/subunit, but measurements give ~ 13 total). Alpha-helix

with Lys, Arg every 3 rd residue.

Countercharges are in other helices.

Note the S4 charge movement, “shots”. Where is the field, precisely? Near the top.

Note the “hinge” in S6, usually a glycine.

Demonstrating the Bezanilla model, #1

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Read the explanation on the simulation.

Show plot. Manual. Then Voltage (start at default, 0 mV ““delayed rectifier”.

Although we simulate sequentially, the cell adds many channels in parallel.

Not an action potential; this is a “voltage jump” or “voltage clamp” experiment.

Describe shots (measure with fluorescence, very approximately).

I = current. Note three types of I.

Describe gating current (average = I(gate); its waveform does not equal the

I(average).

Show -30 mV (delayed openings,) -50 mV (no openings), 0 (default).

Note tail current.

Note I(gate).

There are many V-gated K channels, each with its own V-sens and kinetics.

Demonstrating the Bezanilla model, #2

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Reminder of Lecture 1: Atomic-scale structure of (bacterial) Na+ channels shows that here, too, partial loss of water is important for permeation

Views from the

extracellular solution

(As in Kandel Figure 5-1, Na+ channels select with their side chains)

Views from the

membrane plane

The entire water-like pathway

Payandeh et al, Nature 2011; Zhang et al, Nature 2012

PDB files 4EKW, 4DXW

Voltage-gated Na+ channels, and Blockade by Tetrodotoxin

Payandeh et al, Nature 2011; Zhang et al, Nature 2012;

Animal Na+ channels have an inactivation flap

The pufferfish toxin, from PubChem

PDB files: 4EKW, 4DXW

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Inactivation: a property of all voltage-gated Na+ channelsand of

Some voltage-gated K+ channels

http://nerve.bsd.uchicago.edu/

http://nerve.bsd.uchicago.edu/Na_chan.htm

Site home:

This model is ~ 10 years older than the K+ channel simulation.

Na+ channel has only one subunit, but it has 4 internal repeats(it’s a “pseudo-tetramer”).

The internal repeats resemble an individual K+ subunit. The “P” region differs, governing the ion selectivity.

Orange balls are Na+.Note that the single-channel current (balls inside cell) requires two events: a) All 3 S4 must move up, in response to DV;b) Open flap. When the flap closes, the channel “inactivates”.The flap may be linked to the 4th S4 domain.The synthesized macroscopic current shows a negative peak, then decays.

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Next lecture employs electrical circuits

See also Appendix A in Kandel

Review your material from Phys 1b, practical

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End of Lecture 4

Henry Lester’s Office HoursMonday, Wednesday, Friday

1:15 – 2In / near the Red Door