Notes for Session 2a,2b  (1/19/99 and 1/21/99)

Electrophysiological experiments

Although we will use acetylcholine (ACh) again this week, we will be studying a pathway considerable more complex than the simple ligand-gated channels of last week.  This pathway figured prominantly in the discovery of synaptic transmission, about 1921. We will use muscarinic acetylcholine receptors.  Look here for a diagram of the pathway.

We have expressed the m2 muscarinic acetylcholine receptor and two subunits of G protein-gated inward rectifier K channels (GIRK) in oocytes.  You will be exploring the properties of the channel--mostly inward rectification.

Set up a two-electrode voltage clamp on the oocytes in normal Ringer solution (high-Na).  Then switch to the various solutions containing various K concentrations, with and without ACh.  Your chart recorder traces will look something like panel a.

Take current-voltage relations using the Session 1A protocol in CLAMPEX.  Your subtracted traces will look something like this.

You should also try to write your own protocol.  Make it a ramp, with a single sweep per trial.  Take about 1 second to sweep from +50 mV to -120 mV.

Plot the current-voltage relations for the ACh-activated current at various [K].  You may get this sort of data.

Learning to make RNA

Beginning this week, one group at a time will be learning to synthesize mRNA and to inject it into oocytes.  Group 1 begins this week.  You will be working with Bryan Smith and Cesar Labarca.

Learning to make your own electrodes

It's also time to learn this skill.  Qiau Zhou is in charge.