Application Note

Channel:Cells:
Tools:

CaV3.2
HEK293Patchliner®

Characterization of CaV3.2 (HEK293) onNanion‘s Patchliner®
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were supplied by Millipore, USA

Summary
The gene CACNA1H encodes the α1H subunit of the voltage-gated calcium channel CaV3.2. It belongs to the low

voltage-activated T-type calcium channels. CaV3.2 displays the typical characteristics of the T-type channels:

activation at low depolarization of the membrane andtransient kinetics.
T-type Ca2+ channels are involved in diverse, mainly rhythmic processes like e.g. pacemaking and generation ofthalamocortical rhythms in sleep or epilepsy. CaV3.2 isexpressed in a wide variety of cells. Amongst others it
has been found in kidney, smooth muscle, brain, adrenal

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

and cardiac cells. It seems to be involved in contractionof smooth muscle and the secretion of the adrenal hormones aldosterone and cortisol. Pharmacological blockof T-type channels may lead to new drugs for the treatment of hypertension and epilepsy.
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) to thetest potential for 50 ms before stepping back to holding.
Test potentials were varied between -50 mV and 40 mV in
10 mV increments.

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Figure 2:
Average current- voltage relationship (n = 12). The error bars reflect theS.E.M.

Application Note

Figure 3:Shown are raw current traces from an individual cell expressing CaV 3.2
under control conditions (black) and at increasing concentrations ofmibefradil as indicated.

Figure 4:Shown is the average dose-response curve for mibefadril. Error bars depict the S.E.M..

Figure 2 shows the average current-voltage relationship of
12 cells. The average mean current at -20 mV of all recorded cells was -785 ± 110 pA (n = 12).
In pharmacological experiments the effect of mibefadril
was determined. Figure 3 shows raw current traces of anindividual cell in the absence and presence of increasing
concentrations of the compound. The average dose-response curve is shown in Figure 4.
The voltage dependence of channel inactivation wasalso examined (Fig 5). Current responses of a double
pulse protocol with varying test potentials between thepulses (5 s) was used to determine the half inactivating
potential. Peak current responses to the second pulse
are expressed relative to the response to the first pulse.
This normalized current and plotted against the voltage
applied between the pulses. The activation curve (Fig 5)

Figure 5:
Average activation and inactivation curves for CaV3.2 as determinedfrom a single and double pulse protocol, respectively.

was constructed from a regular IV step protocol (Fig 1).
Both curves in Figure 5 were fitted with the Boltzmann
equation and revealed a half-inactivating potential of-65 mV and a half-activating potential of -33 mV.

Methods
Cells/Cell Culture

Electrophysiology

HEK293 cells stably expressing CaV3.2 were kindly sup-Whole cell patch clamp recordings were conducted ac-plied by Millipore. Cells were cultured and harvested ac-

cording to Nanion’s standard procedure for the Patch-cording to Nanion‘s standard cell culture protocol.

liner®. Currents were elicited using a voltage step from aSolutions

holding potential of -80 mV for 50 ms to different test po-Standard patch clamp solutions were used modified forCa2+ channel recordings. The external solution contained

tentials and back to holding. Pulses were elicited every
10 seconds.

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 run-down.

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