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Bi / CNS 150 Lecture 2

Friday, October 4, 2013

Voltage-gated channels (no action potentials today)

Henry Lester

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

The Bi / CNS 150 2013Home Page

Please note:

Henry Lester’s office hours

Read the book

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If you drop the course,

or if you register late,

please email Teagan Wall

(in addition to the Registrar’s cards).

Also, if you want to change sections,

please email Teagan

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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

(Like Kandel Figure 5-15)

From Lecture 1

Gate

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[neurotransmitter]

openclosed

chemical transmission atsynapses:

electric field

openclosed

electrical transmission inaxons:

actually, E

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_Mead

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Intracellular recording with sharp glass electrodes

V

= RC = 10 ms; too large!

C = 1 F/cm2

E

R = 104 -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 s 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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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, as in Lecture 1, Slide 22.

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 V;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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http://www.krl.caltech.edu/Projects/physicscourses/index.htm

Monday’s lecture employs electrical circuits

See also Appendix A in Kandel

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End of Lecture 3