Updated 3/4/99

Bi/CNS 161 Winter 1999

organizational meeting: Tuesday 1/5 1 PM SHARP
Beckman Institute 60 (Basement, next to Laurent, near Fraser and Koch)

 Start here: email your info to Henry Lester

Description, goals (from the Caltech catalog)

Timing and Schedule conflicts

Which rig is yours?

Instructors and their co-ordinates

Master Schedule of Experiments

Course Requirements

Need help on the concepts or techniques?



Prepare now for Independent Projects (Week of March 2)

From the catalog:

                                 Cellular and Molecular Neurobiology Laboratory.
                                 9 units (0-9-0); second term. Prerequisite: Bi 150 or
                                 Instructor's permission. Experiments on the
                                 molecules of membrane excitability--ion channels,
                                 receptors, and transporters. Students synthesize
                                 mRNA in vitro for these molecules from cDNA clones
                                 and inject the mRNA into Xenopus oocytes. Students
                                 then perform electrophysiological experiments on the
                                 oocytes, including voltage-clamp recording of
                                 macroscopic currents and patch-clamp recording of
                                 single channels. Students analyze the data to reveal
                                 quantitative biophysical concepts. Graded pass/fail.
                                 Given in alternate years; offered 1998/99.. Instructor:

Times: Tuesday and Thursday PM, 1 PM ******sharp****** to 6 PM, 60 Beckman Institute

Please arrive on time 1 PM

Rig assignments






Stephen Shepherd <mailamber@caltech.edu> Jr Bi/CS 1
Svjetlana Miocinovic <sm@ccox.caltech.edu> Jr Bi/CS 1
Kayla Smith <kayla@cco.caltech.edu> Jr Bi 1
Rory Sayres <sayres@its.caltech.edu> Jr Bi 1
Eugene Pivovarov <evgueny@cco.caltech.edu> Gd3 Phys 2
Luis Vazquez <vazquez@its.caltech.edu Gd1 Biol 2
Josh Maurer <jmaurer@its.caltech.edu> Gd3 Chem 2
Ryan Simkovsky <ryans@cco.caltech.edu> So Bi 3
Melinda Turner <mlturner@its.caltech.edu> So E&AS:CNS 3
Brent Kious <kious@its.caltech.edu> Jr Bi/SES 3
G. Bjorn Christianson <bjorn@vis.caltech.edu> Gd1 CNS 4
Ofer Mazor <mazor@its.caltech.edu> Gd1 CNS 4
Ania Mitros <ania@goethe.klab.caltech.edu> Gd1 CNS 4
Javier Perez-Orive <javierpo@caltech.edu> Gd1 CNS 4
Matt Paul <mattpaul@ugcs.caltech.edu> Jr E&AS 5
Gerard Paul Vigil <paulvig@its.caltech.edu> Sr SES 5
Minoree Kohwi <minoree@its.caltech.edu> Sr Bi 5
Urie Eden <eden@cco.caltech.edu> Sr Math/CNS 6
Patrick Drew <notpat@ugcs.caltech.edu> Sr Biol 6
Ethan Snyder-Frey <ethan@ugcs.caltech.edu> Sr Bi/CS 6
Gabriel Miller <gabriel@caltech.edu> Sr Bi/Ch 6

Please arrive on time 1 PM

Instructor: Henry Lester, 328 Kerckhoff, x4946 lester@caltech.edu

 Robert Bao 795-6521
 Marcus Sarofim X6046
grad student
Cesar Labarca X6047
Member of the
Professional Staff
George Shapovalov
grad student
Single channels
 W Bryan Smith
grad student
 Qiao Zhou
grad student

Please arrive on time 1 PM

Help on the concepts and techniques

Please review (make sure you understand the difference between these 2 books):
either the book used in Bi 150 1996: Kandel, et al (Essentials . . .): 7-11 and 16
or Kandel, et al 3rd edition (Principles . . .): Chapters 5 - 14 and Appendix A
(both books are on reserve at the library).
References on techniques are also available on the Library:
Methods in Enzymology,  volume 207 (Ion Chanels, Part A)
Methods in Enzymology,  volume 293 (Ion Chanels, Part B)
Methods in Enzymology,  volume 294 (Ion Chanels, Part C)
Methods in Enzymology,  volume 296 (Neurotransmitter Transporters)

The Axon Guide for Electrophysiology and Biophysics
Note: you don't need to follow this link from the Rig computers.  The Axon Guide is on the Desktop.

Henry Lester's Home Page

Ion Channel Network

Ecitatory Ligand-induced Channels

Master Schedule



cRNA injection Recording technique Data acquisition/




January 12,14
Model oocyte

Mouse AChR abgd subunits 

2-electrode clamp,

"slow" and "fast"

Chart recorder

 (also used for all subsequent weeks)



ACh George S. away
January 19,21 m2-muscarinic receptor; GIRK1+GIRK2 2-electrode clamp




ACh, atropine,


January 26,28 Shaker H4 K+ channel; Shaker IR channel 2-electrode clamp




HAL away Monday (N.Y.)
February 2,4 Na channels, Shaker IR 2-electrode clamp
2-electrode current-clamp recording


Normal Ringer;
10% Na Ringer
2/2 Marcus away
February 9,11
(Note next row also)
More action potentials 2-electrode current-clamp recording CLAMPEX/
 CLAMPFIT, and chart recorder
Normal Ringer 2/9 Henry at a funeral
5HT2C receptors 2-electrode clamp;
fura-2 optical recordings
Chart recorder; 
Axon Imaging Workbench
5-HT 100 nM
ACh receptor-channel blockers
February 16,18 AChR and mutants
Inward rectifiers
patch CLAMPEX/
ACh, QX-222 HAL away all week (Baltimore)
February 23,25 Slowpoke patch CLAMPEX HAL away 

Thursday (D.C.)

March 2,4 Projects: to be arranged

See schedule

To be arranged 
March 9,11 March 9: Project Experiments
March 11:
Project oral reports
To be arranged 
March 16  Notebooks and project reports due.

Please arrive on time 1 PM
Course requirements:

1. Show up - on time - when you are in town and have no conflict with another course.

2. Keep a conscientious lab notebook for your data and analyses.  Submit it on March 16, 9 AM.

3. Give a seminar on March 11.

Independent Projects

"Bright ideas"

Each group should generate a proposal for semi-independent experiments to be performed during the week of March 9. Your proposal should focus on Xenopus oocytes injected with RNA and studied with one of the techniques we will already have used (voltage clamp or patch clamp) or with another technique that you might like to learn. Examples of other techniques would be radioligand binding, tracer flux, or dye measurements of intracellular Ca.

You may reorganize your groups if everyone is in agreement.

Your project should be a real experiment, not a duplication of something we have already done, although you may do something different with an RNA we have previously studied (*). The # sign denotes cRNAs for which site-directed mutations have been made and studied. You might want to design an experiment around such mutants.

A preliminary one-page summary of your experiment should be given or Emailed to H. Lester (Lester@Caltech.edu) by February 15. You should develop your idea yourself, in consultation with Henry, or with other students or T. A.'s. We will then discuss and refine your idea together. A major purpose of this project is to teach you to use the original literature efficiently and intelligently. You may also want to consult the books available on reserve or the information on Henry's web page.

Please note that, although we do in principle have these reagents available, it may take a while to check them out and generate new cDNAs and/or oligos so that you can make RNAs. I'm also willing to phone my friends and beg for some interesting clone that's not on the list. So please allow us to plan effectively by talking with us in advance!

7-helix receptors

Serotonin 5HT1C
Serotonin 5HT1A#
muscarinic M1, M2
Thyrotropin releasing hormone
mu, delta, and kappa opioid

Voltage-gated channels

Shaker IR (inactivation removed)*

Arabidopsis (yes, a plant channel!)

SkM2 cardiac Na channel
Na beta subunit
Cardiac Ca channel

Ca-activated K channel


Inward rectifier channels

GIRK1 through GIRK4 (G protein gated inward rectifier)#

phosphorylation-activated channel

cystic fibrosis transmembrane conductance regulator (CFTR)*#

neurotransmitter-gated channels

Muscle ACh (alpha, beta, gamma, delta subunits)*#
Neuronal alpha2, alpha4, alpha7, alpha8, beta2#, beta4# subunits
NMDA receptor
5HT3 serotonin receptor
P2X2, P2X3, P2X4, P2X7 ATP-gated channels

electrogenic transporters

GAT1 GABA transporter#
GLY1 glycine transporter
Serotonin transporter
Na/glucose transporter

cyclic nucleotide-gated channels

olfactory, subunit 1
olfactory, subunit 2

Tissue-derived mRNA:

Your proposal could be an informed "fishing expedition" to look for interesting responses from these RNAs, which presumably encode proteins expressed by these rat tissues. Your proposal should cite previous studies with these RNAs and suggest a specific experiment.
olfactory epithelium

Campus address
phone contact
Status and option (i.e., grad 3rd year, physics)
Neuroscience experience (Bi 150, etc)
Best afternoons and hours to attend the lab; list conflicts
Level of certainty to take the course (0=no; 1= definitely)

Your own data:
paste this template into your email program,
fill in the blanks, and send it to Henry Lester
Campus address
phone contact
Status and option (i.e., grad 3rd year, physics)
Neuroscience experience (Bi 150, etc)
Best afternoons and hours to attend the lab; list conflicts
Level of certainty to take the course (0=no; 1= definitely)

Rinse the pipette-filling syringes ($20 apiece !!)
rinse solutions from tubing and chamber
empty vaccum reservoir
clean up any 3 M KCl
turn off oscilloscope and Geneclamp
cap on chart pen
oocytes down sink

Glass into disposal boxes