Biology Reference
In-Depth Information
regulatory proteins, even if their expression was upregulated by the cell to com-
pensate for the increased expression of the channels.
Recording the activity of recombinant channels from cells that are dialyzed with
simple salt solutions using the conventional ''whole-cell'' configuration of the
patch clamp is also dangerous because in the absence of normal metabolic activity,
many calcium channels have conformations from which it is di
cult to elicit
robust activity with physiological stimuli. Nevertheless, if the plasmid drives the
expression of 100-10,000 times more channels than are normally present, even
channels with very little activity might generate quite large currents. We would
argue, however, that identifying the mechanisms that halve or double the activity
of recombinant channels, which only open for 1 or 2 ms every second, might not be
relevant to their physiological functions. Thus, this chapter focuses on the more
di
Y
cult and consequently less frequently used techniques of single-channel record-
ing from cell-attached patches and the perforated patch technique for voltage
clamping metabolically intact cells.
Y
II. Rationale
Calcium signaling can be investigated at many levels. Although the patch clamp
method is a very quantitative technique for measuring ion fluxes across the plasma
membrane, internal stores of calcium are important sources that the patch clamp
technique cannot access. To study calcium release from internal stores through
calcium-selective channels, one must use fluorescent calcium indicators or recon-
stitute the channels from organelles in bilayers. This chapter provides an overview
of the patch clamp method for measuring voltage-activated calcium currents
across the plasma membrane. We have applied this technique primarily to dis-
sociated mammalian cells in vitro culture, and it has also been adapted to studying
neurons in brain slices (
Sakmann and Neher, 1995
). Very basically, the technique
involves the use of an operational amplifier circuit to clamp voltage changes
between a wire in the patch pipette and a wire in the bath and the measurement
of how much current it takes to hold the voltage constant. When a giga-ohm
(G
O¼
10
9
ohms) seal is formed between the glass patch pipette and the cell
membrane, background current fluctuations can be reduced su
Y
ciently to detect
picoampere currents. Gigaohm seals are most e
ective for stable, low-noise re-
cording when they are in the 10-100 G
O
range. However, in our experience, most
investigators routinely settle for seals in the 1-10 G
O
range. The methods de-
scribed below allow us to routinely obtain seals around 50 G
O
. Unfortunately,
they are all necessary for success.
When the patch clamp technique was introduced almost 30 years ago (
Hamill
et al., 1981
), it was the only quantitative method available to obtain reliable
physiological information about calcium signals in mammalian cells and the
proteins that mediate them. Now, however, there are increasingly sophisticated
calcium indicators that can be targeted genetically to specific compartments in
V
