Bi / CNS 150 Lecture 2 Friday, October 4, 2013

1. Bi / CNS 150 Lecture 2 Friday, October 4, 2013 Voltage-gated channels (no action potentials today) Henry Lester

2. The Bi / CNS 150 2013 Home Page http://www.cns.caltech.edu/bi150/ Please note: Henry Lester’s office hours Read the book

3. 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

4. H2O K+ ion carbonyl In the “selectivity filter” of most K+ channels, K+ ions lose their waters of hydration and are co-ordinated by backbone carbonyl groups From Lecture 1 Ion selectivity filter Gate (Like Kandel Figure 5-15)

5. [neurotransmitter] chemical transmission at synapses: open closed Future lectures: actually, DE electric field electrical transmission in axons: open closed Major Roles for Ion Channels

6. The electric field across a biological membrane, compared with other electric fields in the modern world 1. A “high-voltage” transmission line 1 megavolt = 106 V. The ceramic insulators have a length of ~ 1 m. The field is ~ 106 V/m. 2. A biological membrane The “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 !!!

7. From Lecture 1 open channel = conductor = Na+ channel

8. Max Delbruck Carver Mead Richard Feynman H. A. L http://en.wikipedia.org/wiki/Carver_Mead 1973

9. V extracellular R = 104W-cm2 C = 1 mF/cm2 E intracellular Intracellular recording with sharp glass electrodes • = RC = 10 ms; too large!

10. A A better way: record the current from channels directly? Feynman’s idea

11. A 5 pA = 104 ions/ms 20 ms A single voltage-gated Na+ channel -20 mV -80 mV Dynamic range 10 ms to 20 min : 108 2 pA to 100 nA 50,000 chans/cell

12. Press release for 1991 Nobel Prize in Physiology or Medicine: http://www.nobel.se/medicine/laureates/1991/press.html

13. Simulation of Shaker gating Francisco Bezanilla's simulation program at the Univ. of Chicago. http://nerve.bsd.uchicago.edu/model/rotmodel.html “Shaker”, a well-studied voltage-gated K+ channel “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).

14. The Hodgkin-Huxley formulation of a neuron membrane 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.

15. Demonstrating the Bezanilla model, #1 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.

16. Demonstrating the Bezanilla model, #2 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.

17. Inactivation: a property of all voltage-gated Na+ channels and of Some voltage-gated K+ channels http://nerve.bsd.uchicago.edu/Na_chan.htm Site home: http://nerve.bsd.uchicago.edu/ • 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: • All 3 S4 must move up, in response to DV; • 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.

18. Monday’s lecture employs electrical circuits http://www.krl.caltech.edu/Projects/physicscourses/index.htm See also Appendix A in Kandel

19. End of Lecture 3