Notes for Session 5b (2/9 and 2/11/99)
Serotonin 5HT2C receptors expressed in oocytes
The transduction
pathway that we study today is the most complex we have used thus far:
5HT2C receptors
G proteins
phospholipase C
IP3 generation
Ca release from intracellular stores
Ca-activated Cl channels
You will study this pathway in 2 ways.
(1) Electropysiologically, as usual, by studying the Cl currents.
Hold the oocyte at -60 mV while you flow in the 5-HT3 (100 nM). Note
that the response occurs within a few seconds, then desensitizes.
Wash out the 5-HT within 2 minutes, wait 10 min, and try again. Which
part of the pathway do you think is desensitiziting? How would you study
this?
(2) Optically, using fura-2 which responds to intracellular calicum. Robert Bao has tuned up this experiment and will dmeonstrate it to you on a special microscope in the Lester lab. Follow him. Fura-2 changes its fluorescence when it binds Ca; see also the general concepts and procedures.