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

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

3. 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. 4 In the “selectivity filter” of most K + channels, K + ions lose their waters of hydration and are co-ordinated by backbone carbonyl groups H2OH2O K + ion carbonyl (Like Kandel Figure 5-15) From Lecture 1 Ion selectivity filter Gate

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

6. 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 = 10 6 V. The ceramic insulators have a length of ~ 1 m. The field is ~ 10 6 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 10 7 V/m !!! Dielectric breakdown fields (V/m) Ceramic8 x 10 7 Silicone Rubber3 x 10 7 Polyvinyl chloride7 x 10 6

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

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

9. 9 Intracellular recording with sharp glass electrodes V  = RC = 10 ms; too large! C = 1  F/cm 2 E R = 10 4  -cm 2 intracellular extracellular

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

11. 11 5 pA = 10 4 ions/ms 20 ms A single voltage-gated Na + channel -80 mV -20 mV A Dynamic range 10  s to 20 min : 10 8 2 pA to 100 nA 50,000 chans/cell

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

13. 13 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

14. 14 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

15. 15 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

16. 16 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

17. 17 Inactivation: a property of all voltage-gated Na + channels and 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 4 th S4 domain. The synthesized macroscopic current shows a negative peak, then decays.

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

19. 19 End of Lecture 3