Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes.
Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-microseconds time resolution, respectively, for studying neurotransmitter receptors in giant (approximately 20-microns diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853-2703, USA.
L
L
Niu
R W
RW
Vazquez
G
G
Nagel
T
T
Friedrich
E
E
Bamberg
R E
RE
Oswald
G P
GP
Hess
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D005260:Female; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010782:Photolysis; D011950:Receptors, Cholinergic / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D014982:Xenopus laevis
D011950:Receptors, Cholinergic; D002217:Carbachol