Application Note

Channel:Cells:
Tools:

CaV2.2
HEK293Port-a-Patch
External Perfusion

Characterization of CaV2.2 (HEK293) onNanion‘s Port-a-PatchTheelectrophysiology

teamatNanion

Technologies

GmbH,Munich.

Cells kindly provided by EMD Millipore, USA.

Summary
The voltage gated N-type calcium channel (Ca­V2.2) is

encoded by the gene CACNA1B. CaV2.2 is a high voltage activated calcium channel.

CaV2.2 is found mainly in the brain, where it mediates

neurotransmitter release at the synapse. The strong depolarization of neuronal action potentials causes theopening of the channel. Calcium can then enter the celland initiates the fusion of the neurotransmitter vesicles
with the membrane.
CaV2.2 is inhibited by w-conotoxin, a neurotoxin of thefish hunting snail, with high specificity. CaV2.2 has been

implicated in the transmission of pain. Pharmacologicalblock of CaV2.2 by compounds based on w-conotoxin

Figure 1:
Representative current responses of an individual cell expressing CaV2.2
to a CaV IV voltage protocol (for details see text).

has been shown to be effective against strong chronicalpain.
The biophysical and pharmacological properties of thecells are presented in this Application Note.

Results
Figure 1 shows current responses of an individual cell toa current-voltage relationship step protocol. Potentials
were stepped from the holding potential (-80 mV) for 50
ms to the test potential before stepping back to holding.
Test potentials were varied between -20 mV and 90 mV in
10 mV increments.
Figure 2:
Average current-voltage relationship (n = 10). The error bars reflect thestandard error of the mean (S.E.M.).

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Figure 3:Shown are raw current traces from an individual cell expressing CaV2.2
under control conditions (black) and at increasing concentrations ofcadmium as indicated.

Figure 4:Shown are raw current responses in the presence of increasing cadmium concentrations as indicated.

Figure 2 shows the average current-voltage relationshipfor ten cells. The average mean current at 30 mV of all recorded cells was -744 ± 140 pA (n = 10).
The inhibition of CaV2.2 by cadmium was studied using theExternal Persusion System. Figure 3 shows the current of anindividual cell at increasing concentrations of cadmium(as indicated). The average dose-response curve is shown
In Figure 4. From this the IC50 was calculated to be 2.7 ±0.4 µM (n = 5).

The inhibition by cadmium was fully reversible, as can beseen in Figure 5. For the cell shown, cadmium was washed
out and applied at its highest concentration another threetimes after collection of a full dose-response curve. Control current levels were recovered each time cadmium

Figure 5:
Block by cadmium is fully reversible. Shown is the timecourse of the peakCav 2.2 current with the application of cadmium as indicated.

was washed out.

Methods
Cell Culture

ElectrophysiologyPrecisION hCav2.2 (α1B/β3/α2δ1)-HEK293 recombinant

Whole cell patch clamp recordings were conducted ac-cell line (CYL3054) was kindly supplied by EMD Millipore,cording to Nanion’s standard procedure for the Port-a-USA. Cells were cultured and harvested according to Na-Patch. Currents were elicited using a voltage step from anion‘s standard cell culture protocol.

holding potential of -80 mV for 50 ms to different test po-Solutions

tentials and back to holding. Pulses were elicited every

Standard patch clamp solutions were modified for the10 seconds.

Ca2+ channel recordings. The external solution contained
20 mM Ba2+ as the charge carrier. The predominant ion
in the internal solution was Cs+ .It also contained Mg-ATP,GTP, and BAPTA to minimize current run down.

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