Detection of intracellular calcium elevations in Xenopus laevis oocytes: aequorin luminescence versus electrophysiology.
Detection of receptor expression in Xenopus oocytes often relies upon functional coupling to second messengers such as Ca2+ or cyclic adenosine monophosphate. To detect intracellular Ca2+, electrophysiological measurement of the endogenous Ca(2+)-activated chloride current (ICl(Ca)) is often used (Dascal, 1987). An alternative utilizes the Ca2+ sensing, bioluminescent protein aequorin (Parker and Miledi(1986) Proc. R. Soc. Lond. B, 228: 307-315; Giladi and Spindel (1991) BioTechniques, 10: 744-747). In the present study the sensitivities of aequorin and electrophysiology for detecting receptor-mediated Ca2+ transients were compared. Assays were performed on the same batches of oocytes using either animal serum or ligands of exogenous receptors to generate inositol 1,4,5-trisphosphate (InsP3) and ultimately elevate intracellular Ca2+. Signal amplitudes were controlled by titrating the concentration of animal serum, or titrating the amount of receptor mRNA injected. Both assays detected signals with high concentrations of animal serum, or with high receptor density. However, aequorin signals were not detected in experiments with average ICl(Ca) current amplitudes below 200 nA. To further evaluate the differences between these two techniques, membrane current and bioluminescence were measured simultaneously. Results of these studies suggest that the signals differ due to the spatial distribution of aequorin, the chloride channels, and the calcium release sites.
Grygorczyk
R
R
Dept. of Biochem and Physiol. Merck Research Laboratories Rahway, NJ 07065-0900, USA.
Feighner
S D
SD
Adam
M
M
Liu
K K
KK
LeCouter
J E
JE
Dashkevicz
M P
MP
Hreniuk
D L
DL
Rydberg
E H
EH
Arena
J P
JP
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D008163:Luminescent Measurements; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D013373:Substance P / Q000494:pharmacology; D014982:Xenopus laevis
D013373:Substance P; D002118:Calcium