1. 1 Bi / CNS 150 Lecture 4 Monday, October 5, 2015 Voltage-gated channels (no action potentials today) Henry Lester

2. 2 http://www.cns.caltech.edu/bi150/ The Bi / NB / CNS 150 2015 Home 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).

3. Ion Channels in the News 5 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 channel Found among many invertebrates Next: PDB file 3RIF

4. 4 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 DNA An older GluCl construct Our 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

5. 5 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 From Lecture 1 Ion selectivity filter Gate (Like Kandel Figure 5-15)

6. 6 [neurotransmitter] open closed chemical transmission at synapses: electric field open closed electrical transmission in axons: actually,  E Major Roles for Ion Channels Future lectures:

7. 7 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

8. 8 open channel= conductor Na + channel = From Lecture 1

9. 9 1973 Max Delbruck Richard Feynman H. A. L Carver Mead http://en.wikipedia.org/wiki /Carver_Meaden.wikipedia.org/wiki /Carver_Mead http://www.nytimes.com/2015/ 09/27/technology/smaller- faster-cheaper-over-the- future-of-computer-chips.html

10. 10 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

11. 11 A better way: record the current from channels directly? Feynman’s idea A

12. 12 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

13. 13 http://www.nobel.se/medicine/laureates/1991/press.html Press release for 1991 Nobel Prize in Physiology or Medicine:

14. 14 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

15. 15 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

16. 16 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

17. 17 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

18. 18 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

19. 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

20. 20 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, 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  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.

21. 21 Next lecture employs electrical circuits See also Appendix A in Kandel Review your material from Phys 1b, practical

22. 22 End of Lecture 4 Henry Lester’s Office Hours Monday, Wednesday, Friday 1:15 – 2 In / near the Red Door