Ion Channels:

Application Note

KCa3.1
Cells:CHO

Modulation of hKCa3.1 by internal Ca2+
performed on Nanion’s Patchliner

Tools:Patchliner

Nanion Technologies GmbH, Munich

Summary
The intermediate-conductance calcium-activated K+ channel,
also known as KCa3.1, IKCa1 or SK4, is a member of the large
family of potassium channels gated by calcium1. It can be

distinguished from the other calcium-activated K+ channels
by differences in channel conductance, calcium sensitivity,
voltage dependence and pharmacological properties1.
The hKCa3.1 channel is encoded by the KCNN4 gene1. It

is primarily expressed in peripheral tissues, including thoseof the hematopoietic system, colon, lung, placenta, andpancreas1 and has been proposed to play an importantrole in a variety of physiological processes including volume
regulation in erythrocytes2, proliferation and differentiation ofB- and T-lymphocytes3,4,5, and tissue protection following spinal
cord injury6. Importantly, the hKCa3.1 channel is a promisingtherapeutic target for a variety of health disorders including
sickle cell anaemia7 and immunological disorders4,5,8. TheCa2+-binding protein, calmodulin (CaM), is required for theactivation of hKCa3.19. The Ca2+-CaM complex is proposed tosolution utilized an optimized procedure on the Patchliner.

ResultsTo compare channel activity in the absence and presence ofinternal Ca2+, exchange of the internal solution is required. The
cells worked well on the Patchliner with a success rate (sealresistance >500 MΩ) of 78% before and 66% after exchangeof the internal solution (n = 32 cells). Currents mediated by
hKCa3.1 were activated by application of 1 µM free-Ca2+ in theinternal solution. Figure 1 shows the current-voltage relationshipof a cell in the absence (black trace) and presence (bluetrace) of 1 µM internal free-Ca2+. The hKCa3.1 current wasreversibly blocked by external application of 5 mM BaCl2 (redtrace). The timecourse of activation of the current followinginternal application of Ca2+ and blockade by external BaCl2,including washout, is shown in Figure 1(top). BaCl2 blocked thecurrent in a reversible and voltage-dependent manner.

bind to an intracellular domain of the C terminus of all subunits
in the tetramer, inducing conformational changes to open thechannel9.
In this study the Patchliner was used to perform a biophysicaland pharmacological characterization of hKCa3.1 channels

0 free Ca2+
1 µM free Ca2+
5 mM BaCl2

expressed in CHO cells. hKCa3.1 was activated by exchanging
the internal solution to a solution containing free-Ca2+ andblocked by external application of non-selective (BaCl2) and

0.5 nA

that reported in the literature4,8. The exchange of the internal

0selective (TRAM-34) inhibitors with an IC50 value consistent with50 ms

1 µM free Ca2+

Current (nA)

1.6

Figure 1: Top: Raw traces from an exemplar cellin the absence (black trace) and presence (bluetrace) of internal calcium and subsequent block by
external BaCl2 (red tace). Bottom: Timecourse of theexperiment.

5 mM
BaCl2

1.2

washINTERNALEXCHANGE

0.8
0.4 0 freeCa2+0.00100

200300 400
Time (s)500600Figure 2: The concentration response curve for
TRAM-34 for an average of 7 cells, revealing an% inhibition

0.6

0.4

0.2

Current (nA)0.8IC50 of 24.3 ± 6.0 nM (n = 7). The timecourse of theTRAM-34 (n = 7)
IC50 = 24.3 ± 6.0 nM
Max = 0.9, Hill = 1.2
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

experiment and traces of an individual cell in theabsence (control) and presence of increasing

5 nM
TRAM-34

concentrations of TRAM-34 are shown in the insets.
50 nM
TRAM-34Control
5 nM
50 nM
300 nM

300 nM
TRAM-34

0

100 200 300 400 500
Time(s)00.5 nA

0.0
100 pM

1 nM

10 nM

50 ms

100 nM[TRAM-34]

To further characterize hKCa3.1 channels the pharmacologicaleffect of TRAM-34 was investigated. TRAM-34 is a membrane
permeable lipophilic compound, designed to be a highly
selective inhibitor of KCa3.1 currents8. Figure 2 shows raw current
traces of an individual cell in the absence (control, 1 µM internal free-Ca2+) and presence of increasing concentrations ofTRAM-34. The timecourse of the experiment is also shown (Figure 2). The average concentration response curve for TRAM-34(Figure 2) reveals an IC50 of 24.3 ± 6.0 nM (n = 7), in excellent
agreement with values reported in the literature4,8.

In conclusion, the Patchliner, in combination with hKCa3.1
expressing CHO cell line (Charles River), is a robustelectrophysiological assay for activation by internal solution
exchange and subsequent block of hKCa3.1 for drug discovery.

References
1. Wei, A.D., et al., 2005. Pharmacol. Rev. 57(4): 463–472.
2. Gardos, G., 1958. Biochim. Biophys. Acta 30: 653-654
3. Khanna, R., et al. 1999. JBC 274: 14838-14849.

Methods
Cells
CHO cells stably expressing KCa3.1 were used, kindly providedby Charles River.

Electrophysiology measurements
Cells were cultured and harvested according to Nanion’s
standard protocols. Cells were resuspended in external
recording solution and stored in the CellHotel of the Patchliner
before being dispensed into each well of the NPC-16 chip.
Internal and external solution compositions are available uponrequest. Currents were elicited using a voltage ramp protocol
from -140 to +60 mV over 200 ms, interval 10 s. Intersweep
holding potential was -100 mV. To activate hKCa3.1 currents,
the chip wagon was removed from the measurehead, theinternal solution exchanged and the recording continued.
an optimized protocol was used for this procedure. The
compounds BaCl2 and TRAM-34 were applied via the external

solution. Cells and solutions were stored at 15°C throughout theexperiments, using the Patchliner CoolingPlate. This improves
cell quality thus extending duration of the recording and cellviability.

4. Wulff, H., et al., 2004. J. Immunol. 173: 776-786.
5. Lam, J. & Wulff, H., 2011. Drug. Dev. Res. 72: 573-584.
6. Bouhy, D., et al., 2011. J. Neurosci. 31: 16298-16308.
7. Brugnara, C., et al., 1996. J. Clin. Invest. 97: 1227-1234.
8. Wulff, H., et al., 2000. PNAS. 97: 8151-8156.
9. Fanger, C.M., et al., 1999. JBC. 274: 5746–5754.

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