Functional Site-Directed Fluorometry 61
Next, a single labeled oocyte is placed in the electrophysiological recording
chamber and its membrane potential controlled by voltage-clamp (Fig. 1c). Simul-
taneously, the covalently attached fluorophores are excited by a light source, and the
resulting excitation is measured with a photomultiplier tube, photodiode, or camera.
Traditionally, excitation sources were broadband sources, such as halogen-tungsten
or low-noise mercury or xenon arc lamps; recently however, LEDs and lasers have
become more practical for use. Their narrower excitation spectra are useful in en-
suring stray excitation light is not transmitted as emission with fewer filter require-
ments. Furthermore, their power levels are often higher than those obtained from
broadband sources, particularly in the shorter wavelengths of the visible spectrum.
Both two-electrode voltage clamp and cut-open oocyte voltage clamp are com-
monly used in site-directed fluorometry experiments to provide a simultaneous
functional assay of the membrane protein. While two-electrode voltage clamp can
be less technically difficult to perform, the cut-open oocyte technique offers some
advantages including a faster clamp to more easily resolve gating currents and an
ability to exchange the internal solution. Regarding site-directed fluorometry in par-
ticular, when using the cut-open oocyte clamp technique the channels measured
optically are identical to those measured electrophysiologically, whereas when us-
ing two-electrode voltage-clamp the channels measured optically are a subset of
those measured electrophysiologically. Thus, comparisons of kinetics, such as of
gating current activation to changes in fluorescence, are typically more accurately
obtained using the cut-open technique. Regardless of which technique is chosen, the
oocyte should be placed so the dark animal pole is facing the light detector in order
to minimize background fluorescence. Oocyte background fluorescence decreases
undergo site-directed mutagenesis via polymerase chain reaction, resulting in ( 2 ) the production of
plasmids containing the mutation. Following transformation and DNA purification from selected
bacterial colonies, the DNA is linearized and used for ( 3 ) RNA ( black, single strand) synthesis
using an in vitro transcription kit. The purified RNA contains the mutation of interest and can
be easily diluted to an appropriate concentration and ( 4 ) injected into oocytes for subsequent
protein expression and site-directed fluorometry experiments. b Fluorophore conjugation. After a
few days of incubation, oocytes express the protein ( yellow oval) on the membrane surface ( dark
green) and the dye ( Fluor) is reacted to the mutated cysteine ( right panel), in this example via
a maleimide reaction. c Schematic of cut-open oocyte functional site-directed fluorometry tech-
nique. The oocyte (a) is placed in the cut-open voltage-clamp setup, which consists of an upper,
middle, and inner chamber. The inner chamber (b) contains the vegetal pole of the oocyte, which
is permeabilized to provide direct electrical access to the inside of the oocyte. The middle chamber
(c) behaves as an electronic guard. The upper chamber (d) contains the animal pole of the oocyte,
from which fluorescence changes and ion channel currents (Im) are simultaneously measured.
Following impalement with the microelectrode (e) used to establish virtual ground within the
oocyte, a water-immersed epifluorescence microscope objective ( f ) is placed with the surface of
the oocyte in focus. Excitation light, here provided by a light-emitting diode (g) is filtered (h), and
reflected off a dichroic mirror (i) to excite a fluorophore conjugated to protein expressed on the
surface of the oocyte. Emission light from the fluorophore passes through the dichroic mirror and
is filtered ( j ) and collected by a photodiode (k) (or photomultiplier) that is attached to a low-noise
current to voltage converter headstage (l ), such as a patch clamp amplifier, to provide a readout of
the change in fluorescence ( Fluo)