Biomedical Engineering Reference
In-Depth Information
opening and closing (gating) of most ion channels is affected by the membrane po-
tential. The voltage clamp method is widely used to study ionic currents in neurons
and other cells. However, the errors in the voltage clamp experiment are due to the
current flowing across the voltage-clamped membrane, which generates a drop in
the resistance potential in series with the membrane, and thus causes an error in
control of the potential. This error, which may be significant, can only be partially
corrected by electronic compensation. All microelectrodes act as capacitors as well
as resistors and are nonlinear in behavior. Subsequent correction by computation is
difficult for membrane potentials for which the conductance parameters are volt-
age dependent.
A recent version of voltage-clamp experiment is the
patch clamp technique
.
This method has a resolution high enough to measure electrical currents flow-
ing through single ion channels, which are in the range of pico-amperes (10
−12
A). Patch-clamp refers to the technique of using a blunt pipette to isolate a patch
of membrane. The patch-clamp technique was developed by German biophysicist
Erwin Neher and German physiologist Bert Sakmann to be able to study the ion
currents through single ion channels. The principle of the patch-clamp technique
is that a small patch of a cell membrane is electrically isolated by the thin tip of a
glass pipette (0.5-1-
m diameter), which is pressed towards the membrane surface.
By application of a small negative pressure, a very tight seal between the membrane
and the pipette is obtained. The resistance between the two surfaces is in the range
of 10
9
ohms (G ohm, which is then called the giga-seal). Most techniques for moni-
toring whole-cell membrane capacitance work by applying a voltage stimulus via a
patch pipette and measuring the resulting currents. Because of the tightness of this
seal, different recording configurations can be made.
Patch-clamp technique measurements reveal that each channel is either fully
open or fully closed (i.e., facilitated diffusion through a single channel is all or
none). Although the initial methods applied a voltage step (similar to Problem 3.7)
and analyzed the exponential current decay in the time domain, the most popular
methods use sinusoidal voltage stimulation. Patch-clamp technique is also used to
study exocytosis and endocytosis from single cells. Nevertheless, cell-attached and
excised-patch methods suffer from uncertainty in the area of the membrane patch,
which is also likely to be variable from patch to patch, even with consistent pipette
geometry. The number of ion channels per patch is another source of variability,
in particular for channels that are present at low density. There is also a concern
that patch formation and excision may alter channel properties. Thus, conductance
density estimates obtained from patches are not very reliable.
Another way to observe the electrical activity of a cell is through
impedance
measurements
, which are measured after applying an alternating current. Imped-
ance (
Z
) is a quantity relating voltage to current, and is dependent on both the
capacitative and resistive qualities of the membrane. The units of impedance are
ohms. In the series pathway, two or more resistors and capacitors are equal to im-
pedance as the vector sum of their individual resistance and reactance.
μ
2
2
2
ZRR
=+
(3.25)
c
