Application Note

Channel:Cells:
Tools:

KV1.3
JurkatPort-a-Patch®
Internal Perfusion

Automated internal perfusion of Jurkat cells
expressing KV1.3 channels
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

KV1.3 is a voltage-gated potassium channel which plays aCurrent responses of an individual cell to 200 ms voltage

role in human T cell activation and proliferation, cell-me-pulses (+40 mV) in the presence of K+ and subsequent

diated cytotoxicity and volume regulation. It is therefore

block by internal perfusion of Cs+ ions are shown in Figure

an important target for the therapeutic control of T cell

1. The block of KV1.3 currents by Cs+ using the internal ex-responses. The KV1.3 channel is endogenously expressedin Jurkat cells, an immortalised T lymphocyte cell line. Thechange device was rapid and complete, and could be
easily reversed by removal of Cs+.

channel is activated by membrane depolarisation at voltages typically more positive than -40 mV with rapid activation kinetics and slower inactivation.
In common with many other voltage-gated potassium

ControlCs-containing internalRecovery

channels, KV1.3 can be blocked by replacing K+ with Cs+
ions in the pipette solution. Using Nanion’s Internal Perfu-sion System, we were able to record stable K­V1.3 currents

from Jurkat cells and reliably block this potassium current
by perfusing the internal side of the chip with a Cs+-containing solution. The KV1.3 mediated current could also becompletely recovered by perfusing the inside of the chipwith K+-containing solution.
KV1.3 mediated currents can also be blocked by internal

perfusion with tetraethylammonium (TEA). We present
data here showing block of KV1.3 by increasing concentrations of TEA using the Internal Perfusion System. The IC50

that we obtained for TEA applied internally was in goodagreement with the literature.

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200 pA
50 ms

Figure 1:
Example traces for KV1.3 mediated currents recorded from a Jurkat cellin the presence of K+ and the subsequent block with internal Cs+. KV1.3
currents were blocked by the internal perfusion of Cs+ and the current
could be completely recovered by replacement back to K+.

Application NoteFigure 2 shows the time course of the peak KV1.3 current in

500 pAthe presence of K+ or Cs+ in the internal solution. The recording using the internal solution exchange device was
highly stable where the internal solution could be ex-50 mschanged from K+ to Cs+ over 19 times, with full recovery
upon return to K+‑containing solution. In this example, therecording lasted over 35 minutes.
KV1.3 mediated currents recorded from Jurkat cells could

also be blocked by increasing concentrations of TEA onControl
0.1 mM TEA
1 mM TEA
10 mM TEA

the internal side. Figure 3 shows traces recorded from asingle cell in control conditions and subsequent block byPeak Amplitude (nA)

increasing concentrations of TEA in the internal solution.

Figure 3:
Example traces for KV1.3 mediated currents recorded from a Jurkat cellin the absence and presence of increasing concentrations of internal
TEA. KV1.3 mediated currents were blocked by the internal perfusion ofTEA in a concentration dependent manner.

1.4
1.2
1.0
0.8
0.6

The estimated IC50 value for TEA was 0.9 ± 0.3 mM (n = 3)

0.4

which is in good agreement with that found in the litera-0.2ture (0.63 ± 0.18 mM; Aiyer et al., 1994).
0510152025
Peak Amplitude (nA)

3035Figure 2:
Time course of the peak KV1.3 current in a single experiment in the presence of K+ or Cs+ in the internal solution. The first exchange is indicatedby the blue/grey circles (see Figure 1). Exchange to Cs+ was possible
over 19 times with full recovery and the recording lasted over 35 min.

In conclusion, the internal exchange device from Nanion
can be used for the relatively rapid administration of ionsand small molecules to the internal surface of the cellmembrane. Recordings using the internal solution exchange device on the Port-a-Patch® are stable and fullexchange can be achieved within a few seconds.

References

Electrophysiology

1. Aiyar, J., Nguyen, A.N., Chandy, K.G., and Grissmer, S.

Whole cell patch clamp recordings were conducted ac-1994. The P-Region and S6 of Kv3.1 contribute to the for-cording to Nanion’s standard procedure for the Port-a-mation of the ion conduction pathway. Biophys. J. 67:

Patch®. Currents were elicited using a voltage step from2261-2264

a holding potential of -80 mV to +40 mV for 200 ms every

Methods

20 seconds. Nanion’s Internal Perfusion System was usedCells

standard internal solution containing K+, Cs+ or TEA.

to constantly perfuse the inside surface of the chip with

Jurkat cells endogenously expressing KV1.3 were used.
Cell culture

TEA was made as a 1M stock solution in water and diluted
in standard K+-containing internal solution accordingly.

Cells were cultured and harvested according to Nanion‘sstandard cell culture protocol.

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