Biology Reference
In-Depth Information
specific cells. In addition, genome cloning, fluorescence confocal microscopy, and
structural studies of membrane proteins have advanced to the point where they are
better suited than the patch clamp technique for identifying which channel proteins
are expressed by specific cell types, how their distribution on and tra
Y
cking to and
from the cell surface is regulated, and their three-dimensional molecular structures.
Nevertheless, the patch clamp remains the technique of choice for investigating the
channels' physiological function and regulation at the molecular level in real time.
In the future, genetic manipulation of channel sequence and expression in model
organisms and mass spectrometry of posttranslational modifications might begin
to supplant even these applications of the patch clamp technique.
Three volumes of Methods in Enzymology have already been devoted to the
patch clamp method, so it would be impossible to provide a su
ciently detailed
introduction here that would allow a novice to make recordings without consulting
other sources. Scientists with little training in physics and/or membrane physiology
should start with the ion channel primer by
Aidley and Stanfield (1996)
and the
technical primers by
Molleman (2003) and Ogden (1987)
. More experienced phy-
siologists could move directly to the compendia by
Sakmann and Neher (1995)
and
by
Rudy and Iverson (1992)
. More specialized topics are covered in subsequent
volumes edited by
Conn (1998, 1999)
. If you are creating your own rig from
scratch, chapter by Jim Rae and Rick Levis in volume 207 is essential reading.
Y
III. Methods
A. Assembling the Patch Clamp Rig
To localize contributions to noise, it is important to assemble the rig slowly,
piece by piece. There are two general classes of noise to eliminate initially. Sinu-
soidal fluctuations arise from electronic interference by power sources and from
mechanical vibrations. In practice, they are often di
cult to distinguish because
they produce oscillations of similar frequency, but both should be eliminated
completely. Later, after giga-ohm seal formation, any remaining noise should
have much higher frequencies. Some of this noise arises from capacitative coupling
between the glass wall of the pipette and the salt solution surrounding it in the
bath, which can be minimized experimentally. Remaining contributions to high
frequency noise can only be reduced by filtering the signal, which limits how brief
an event of a given amplitude one can detect.
To begin, run the calibration tests that are specified by the manufacturer on the
amplifier while it is sitting on a desk. Then assemble the air table, the faraday cage,
the microscope, and the manipulator. They should share a common ground. The
lamp of the microscope will require a DC power supply and each of the manip-
ulator's motors must be grounded separately. Surprisingly, despite all their metal
parts, many microscopes are not isopotential and need to be grounded at several
points. With the input of the amplifier's headstage still open and the output filtered
Y
