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Application Note

Kv1.3
Sf21 and Effector Memory T cells
Patchliner®

Pharmacological analysis of heterologous and endogenous expressed
Kv1.3 channels in Sf21 insect cells and T-lymphocytes
Author: Maike Hellmers, M.Sc., Electrophysiologist, conoGenetix biosciences GmbH, Munich.

SummaryResults

The voltage-gated potassium channel Kv1.3 isexpressed in human naive, central memory T cells(TCM), and effector memory T cells (TEM). TEM cells
are important mediators of a variety of chronic
inflammatory autoimmune diseases including
multiple sclerosis (MS), rheumatoid arthritis (RA),psoriasis, and vasculitis. Antigenic stimulation of
TEM cells results in upregulated Kv1.3 channel
expression.

For the comparison of characteristic voltage
dependent Kv1.3 channel activation a typicalvoltage protocol from -60 mV to +60 mV in 20 mV
steps (activation pulse 200 ms) was used. Cells
were clamped at a holding potential of -100 mV.

It is proposed that a selective block of Kv1.3
channels leads to an inhibition of functionality of
TEM cells without affecting the function of naive
and TCM cells. Thus, Kv1.3 is a promisingtherapeutic target that could be used toefficiently medicate chronic autoimmune diseases
without generating typical side effects associatedwith today’s immunosuppressive therapies.

Figure 1 shows the current response of two
individual Sf21 and TEM cells (figure 1A and 1B,respectively) and the pulse protocol for voltage
dependent Kv1.3 activation (1C).
A) Sf21 cell

200 pA

20 ms

0.00

In this Application Note we compare theelectrophysiological properties of the potassium
selective ion channel Kv1.3 expressed in Sf21 cells
and endogenously expressed Kv1.3 channels inTEM cells. For the pharmacological analysis the4 - channel Nanion Patchliner® and one of ourinnovative, Kv1.3 specific, proprietary peptides
was used.

0.02

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0.16

0.18

0.20

0.22

0.24sB) TEM cell
500

400

300

100 pApA

In the laboratory of conoGenetix biosciences theinsect cell line Spodoptera frugiperda (Sf21) isused in combination with a Baculovirus infection
system for the heterologous expression ofrecombinant ion channels. Sf21 cells offer highly
specific protein expression and correct
posttranslational modifications resulting infunctionalchannels
withcomparable
electrophysiological parameters of ion channels invivo.

20020 ms

1000-100
0.00

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0.220.24sC) Pulse Protocol-60 mV-40 mV-20 mV-20 mV-20 mV-40 mV-60 mV

10 ms

200 ms50 ms-100 mV

Figure 1:
Representative voltage dependent activation of Kv1.3 channels
expressed in Sf21 cells (A) and endogenously expressed in TEM
cells (B). (C) Pulse protocol for voltage dependent Kv1.3activation.

Page 1 of 4

Channel:
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Application Note

4.0

2.5

-93.0

It is postulated that quiescent human peripheral
blood naive, TCM, and TEM cells have similar
channel phenotypes expressing ~300 voltagegated Kv1.3 potassium channels per cell (Matheuet al., 2008). The patch clamp analysis displayedthat purified and anti CD3 activated TEM cells
showed an average maximal current of 290 pA, aconductance of 4,8 nS and consequently acalculated number of 370 channels per cell (meanvalues from n=35 cells).
One should note that the calculations are madeon the presumption that all present Kv1.3
channels are activated and opened by the applied
depolarization. In addition the conductance of thecell membrane and subconductances were nottaken into account.

3.5

x10

Normalized Current [nA]

Figure 2 shows the corresponding normalized
voltage-current-correlation. Kv1.3 starts to open
at a depolarization of the membrane from theholding potential (-100 mV) to -20 mV andreaches its maximum open state at +60 mV. Even
though the expression of Kv1.3 is much stronger
in Sf21 cells, the characteristic voltage dependent
activation remains unchanged.

2.0

1.5
1.00.5-60-40-20-0.5

Kv1.3
Sf21 and Effector Memory T cells
Patchliner®

204060

Voltage [mV]Figure 2:
Characteristic normalized voltage-current-correlation ofKv1.3 channels expressed in Sf21 (dark-blue and light-bluelines) and TEM cells (black line); n= 41, n= 12 and n=29,
respectively. With the Sf21 Baculovirus expression system it
is possible to express high amounts of Kv1.3 potassiumchannels. According to the infection rate Sf21 cells can adapt
different expression levels.

The maximal observed current, which clearlyoriginates from Kv1.3 activity, and the applied
voltage allow to assess the total conductivity of all
Kv1.3 channels. Accordingly the number ofchannels can be extracted using the known
conductivity of a single Kv1.3 channel (13 pS;
Grissmer et al., 1990). The maximal K+ outward
current is defined as the difference between
activated unblocked and maximally blocked (fullblock) currents during the first 20 % of the current
trace. Due to the infection conditions, it ispossible to control the expression level of Kv1.3 in
Sf21 cells, according to the needs of theinvestigation. Under our standard conditions amaximal current of 2 - 3 nA, a conductance of33 - 50 nS and a number of 2500 - 3800 channels
per cell can be estimated.
Page 2 of 4

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Application Note
The highly specific Kv1.3 block was performed
with a new and proprietary peptide compounddesigned by conoGenetix. The peptide shows highaffinity to Kv1.3 channels as shown in figure 4.

Kv1.3
Sf21 and Effector Memory T cells
Patchliner®

As shown in figure 5 the averaged IC50 value ofthe peptide for Kv1.3 channels expressed in Sf21
and TEM cells is in good agreement with91,1 nM ± 4,0 nM on Sf21 and 92,8 nM ± 2,3 nM
on TEM cells.

A) Sf21 cell
1.0control
50 nM
125 nM
250 nM
2 µM

2.5nA

2.0

1.5

1.0

500 pA

Relative inhibition

3.0

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0.40.34

0.0

3.5

NormalizednACurrent [nA]

0.6

0.2

0.0

100 pM3.0
2.0

100 nM

1 µM

Figure 5:
Dose response curves of the specific peptide on Kv1.3channels. Blue line Sf21 cells, IC50 = 91,1 nM ± 4,0 nM (n=4);
black line TEM cells, IC50 = 92,8 nM ± 2,3 nM (n=4).

1.5

1.0
0.5
0.0
0.00

Conclusion

time[s]B) TEM cell
500control
50 nM
125 nM
250 nM
2 µM

400

300

200

100 pA
10020 ms

0-100
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0.24

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0.34

0.5
0.4
0.3
0.2
0.1
0.0

10 nM

Concentration

2.50.00

1 nM

CgTX
CgTXSingle
SinglePoint:
Point:Block
Block@@2e-06M
2e-06M==0.68
0.71±±0.07
0.058
(n=4)
(n=3)
CgTX
CgTXIC50:
IC50:Max=0.68±0.072,
Max=0.74±0.05, KD=92.8nM±2.3nM,
KD=91.1nM±4.0nM,Hill=1.2±0.16
Hill=1.5±0.13(3(4fitsfitsaveraged)
averaged)005n
50n 125n 250n 500n 2µCgTX 544
CgTX 544
CgTX 544
CgTX 544
CgTX 544
CgTX 544
CgTX 544

pA

KD Range: 89.0nM
94.9nM - 91.6nM
89.5nM(fit through avaraged values)

0.8

20 ms

0.5

NormalizednACurrent [nA]CgTX_544 (n=4)(n=3)
Max=0.7, KD=90.1nM,
KD=92.3nM, Hill=1.5
Hill=1.2

time[s] 0.00

005n50n 125n 250n 500n 2µCgTX
CgTX 544
CgTX 544
CgTX 544
CgTX 544
CgTX 544
Figure4: 544 CgTX 544

Block of Kv1.3 channels expressed in Sf21 (A) and TEM (B)cells. Representative raw current traces and time dose
responses in the presence of increasing concentrations of theKv1.3 antagonistic peptide.

The heterologous expression system in Sf21 cells
displays a promising method for electrophysiological analysis of pharmacologicallyinteresting ion channels in vitro. In our laboratory
we were already able to overexpress different
potassium channels of the Kv1 subfamily in Sf21cells. Based on our observations it can beassumed that the folding and insertion of Kv1.3
ion channels into the plasma membrane of Sf21
cells is comparable to native proteins in TEM cells.
We
have
clearlydemonstrated
that
heterologously expressed Kv1.3 channels canserve as a model system to study drug targetingby assessment of electrophysiological parameters. This established model system would
allow to furthermore investigate the effect of e.g.
channel mutants and expression levels on drug
effectiveness.

Page 3 of 4Channel:
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Application Note
Methods
Cells
The Spodoptera frugiperda (Sf21) insect cell line
was purchased from Invitrogen.
Untouched TEM cells from quiescent humanperipheral blood were purified using RosetteSeparation and StemCell Negative Selection
columns.
Cell culture
Sf21 suspension roller cultures were cultured,infected and harvested according to theconoGenetix biosciences standard cell culture
protocols.
Purified TEM cells were cultured according to our
specific cultivation protocols and Kv1.3 expression
was induced with anti CD3 activation.

Kv1.3
Sf21 and Effector Memory T cells
Patchliner®

For pharmacological investigations the Kv1.3
currents were elicited using a voltage step fromthe holding potential (-100 mV) to +60 mV for
200 ms and back to the holding potential. Voltage
protocols were elicited every 10 s (KvIV) and 20 s(KvPharm).
The detected currents were pre-amplified onheadstages and digitzed, using a HEKA quadro
patch clamp amplifierer EPC10. Data recroding
was conducted using the PatchMaster software(HEKA Elektronik Dr. Schulze GmbH, Lambrecht /Pfalz, Germany). Data analysis and presentation
was realized using the softwares IGOR Pro Version
6.22A (Wave Metrics Inc., USA) and Microsoft
Excel 2010 (Microsoft Corporation, USA).
Please contact us for further information.

References
1. Grissmer, S., Dethlefs, B., Wasmuth, J. J.,
Goldin, A. L., Gutman, G. A., Cahalan, M.
D., Chandy K. G. (1990). Proc. Natl. Acad.
Sci USA, 87, 9411-9415

Electrophysiology
Whole cell patch clamp recordings wereperformed according to our standard patch clamp
protocols with the 4 - channel Nanion Patchliner®.
Due to the diameter of the cells for Sf21 cells a2-3 MΩ resistance, for TEM cells a 5-6 MΩresistance NPC-16 chip was used. The patch clamp
solutions were adjusted to the Kv1.3 potassiumchannel and the used cell line. The pulse protocol
for the comparison of the voltage-currentcorrelation was identical for both cell lines (seefigure 1C).

2. Matheu, M. P., Beeton, C., Garcia, A., Chi,V., Rangaraju, S., Safrina, O., Monaghan,K., Uemura., M. I., Li, D., Sukumar, P., dela Maza., L. M., Monuki, E., Flügel, A., .
Pennington, M. W., Parker, I., Chandy, G.,Cahalan, M. D. (2008). Immunity, 29, 602–614

conoGenetix biosciences GmbH
Dr. Andreas Klostermann, CEOAm Klopferspitz 1982152 Planegg OT Martinsried

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