Application Note

Channel:Cells:
Tools:

CaV2.2
HEK293Patchliner

Characterization of CaV2.2 (HEK293) onNanion‘s PatchlinerTheelectrophysiology

teamatNanion

Technologies

GmbH,Munich.

Cells kindly provided by Millipore, USA.

SummaryResults

The voltage gated N-type calcium channel (Ca­V2.2) isFigure 1 shows current responses of an individual cell toage activated calcium channel.

were stepped from the holding potential (-80 mV) to theencoded by the gene CACNA1B. CaV2.2 is a high volt-a current-voltage relationship step protocol. Potentials
test potential for 50 ms before stepping back to holding.

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

Test potentials were varied between -20 mV and 90 mV inneurotransmitter release at the synapse. The strong de-

10 mV increments.

polarization of neuronal action potentials causes theFigure 2 shows the average current-voltage relationshipopening of the channel. Calcium can then enter the cellof ten cells. The average peak current at 30 mV of all re-and initiates the fusion of the neurotransmitter vesicles

corded cells was -698 ± 115 pA (n = 6).

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

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

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

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Figure 2:
Average current- voltage relationship (n = 10). The error bars
reflect the S.E.M..

Figure 4:
Average cadmium dose-response curve. Error bars represent
the S.E.M.

Figure 3 shows the current response of an individual cell in
the presence of increasing cadmium concentrations. The
average dose-response curve for block of CaV2.2 by cadmium is shown in Figure 4. The IC50 was calculated from its
Hill fit to be 3.6 ± 0.4 µM (n = 5).

Given the importance of CaV2.2 in pain pathways and in
the treatment of chronic pain, a planar patch clamp de-vice with increased throughput is an invaluable tool tostudy this target. The Patchliner‘s low compound conFigure 3:
Shown are raw current traces from an individual cell expressing CaV2.2 under control conditions (black) and at increasing
concentrations of cadmium (as indicated).

sumption is just one of its critical features that makes it theperfect tool for the CaV2.2 target where low volume toxins
are likely drug candidates.

Methods

Electrophysiology

Cells

Whole cell patch clamp recordings were conducted ac-PrecisION hCav2.2 (α1B/β3/α2δ1)-HEK293 recombinant
cell line (CYL3054) was kindly supplied by EMD Millipore,USA.

cording to Nanion’s standard procedure for the Patchliner®. Currents were elicited using a voltage step from aholding potential of -80 mV for 50 ms to different test potentials and back to holding. Pulses were elicited every

Cell culture

10 seconds.

Cells were cultured and harvested according to Nanion‘s standard cell culture protocol.
SolutionsStandard patch clamp solutions were modified for theCa2+ channel measurements. 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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