Application NoteChannel:
Cells:
Tools:

CaV3.2
HEK293Port-a-Patch®
External Perfusion

Characterization of CaV3.2 (HEK293) onNanion‘s Port-a-Patch®
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

Figure 1:
Representative current responses of an individual cell expressing CaV3.2
to a CaV IV voltage protocol (for details see text).

has been found in kidney, smooth muscle, brain, adrenal
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.
This cell has no considerable tail current. It should be noted, though, that some cells showed bigger tails currents,
as e.g. the cell shown in Figure. 3.

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Figure 2:
Average current-voltage relationship (n = 11). The error bars reflect theS.E.M..

Application NoteFigure 3:
Shown are raw current traces from an individual cell expressing Cav 3.2
under control conditions (darkest blue) and at increasing concentrations of mibefradil as indicated.

Figure 4:Shown are average dose-response curves for the T-type Ca2+ channel
blockers, as indicated.

Figure 2 shows the average current-voltage relationship ofeleven cells. The average mean current at -20 mV of allrecorded cells was -1.4 ± 0.1 nA (n = 10).
Dose-response curves were determined for two Ca2+ Ttype channel blockers: mibefadril and amiloride. To do sothe External Perfusion System was used. Figure 3 shows exemplary raw current traces of an individual cell in presence of increasing concentrations of mibefadril. Similar
experiments were conducted with amiloride. Average
dose-response curves for both compounds are shown inFigure 4.
The voltage dependence of channel inactivation wasalso examined (Figure 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 was plotted against the voltage

Figure 5:
Average activation and inactivation curves for Cav3.2 as determinedfrom a single and double pulse protocol, respectively.

applied between the pulses. The activation curve (alsoFigure 5) was constructed from a regular IV step protocol(Figure 1). Both curves in Figure 5 were fitted with theBoltzmann equation and revealed a half-inactivating
potential of -64 mV (n = 5) and a half-activating potentialof -32 mV (n = 11).

Methods
Electrophysiology

Cells/Cell Culture
HEK293 cells stably expressing CaV3.2 were supplied byMillipore. Cells were cultured and harvested according
to Nanion‘s standard cell culture protocol.
Solutions

Whole cell patch clamp recordings were conducted according to Nanion’s standard procedure for the Port-aPatch®. Currents were elicited using voltage steps from aholding potential of -80 mV for 50 ms to different test potentials and back to holding. Pulses were elicited every

Standard patch clamp solutions were modified for the

10 seconds.

Ca2+ channel recordings. 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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