Multichannel Systems HiClamp Manual - Manual (Page 42)

HiClamp Manual
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5.7.2 The concentration activation curve
A first example of a program sequence is the determination of the concentration activation curve
for a ligand gated channel. To establish such relationship the response of a cell exposed to a series
of ligand concentration is measured and the concentration activation curve is obtained by plotting
the peak of the evoked current (or, in some cases, the area under the curve) as a function of the
logarithm of the agonist concentration. A sequence typically used for these measurements
is illustrated in the next screenshot.
In step 1, the oocytes are automatically loaded from the loading plate (feeder). Oocytes
displaying a membrane potential less than -15 mV are automatically rejected. Following
a successful recording of the membrane potential the leak current is then determined in step 2
(10 s recording). Step 3 determines if the holding current satisfies the desired conditions (typically
less than -1
A). Step 4 and 5 are used to assess the cell response and cells displaying a response
superior to 200 nA are retained. The concentration activation curve is then measured between
steps 7 to 11. Note the red lines indicating the logical steps of the “if” whereas blue lines indicate
the logical steps of the loop. Note also the difference between the two loops (with and without
increment respectively). The sequence terminates once the desired number of cells is measured
(here 8) or the number of available oocytes is reached. The icon end indicates that the program
stops, unload the remaining oocyte and closes the perfusion.
The program sequence is established either by inserting the icons in the proper sequence,
by a single click on the icon tool bar, or by loading a predefined sequence. Loading of a
predefined sequence can be done either using menu File ”Open” to open a file sequence
or by a single click on a “Super Icon” displayed in the upper part of the window.
5.7.3 Recording Voltage-Gated Channel activity
Although analysis of ligand-gated and voltage-gated channels share significant similitude
experimental paradigm significantly differ. While in the case of ligand-gated channels
measurements generally start before moving the cell into the well containing the solution
of interest recordings of voltage gated channel activity can be quite different as we shall
illustrate in the next section.
The voltage protocols
Voltage protocols used for the activation, inactivation or characterization
of voltage-gated channels include single steps, multiple steps or ramps.
Voltage steps are programmed in the voltage command window. In the
simplest case, lets consider that the sequence comprises the following
elements: