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\subsection*{DmCa\textsubscript{v}3 channel produces LVA currents in \emph{Xenopus} oocytes}
To determine biophysical properties of We cloned a full length fly T-type Ca\textsuperscript{2+} channel
(DmCa\textsubscript{v}3), we first cloned the full length cDNA
clone by assembling fragmented PCR products amplified (named DmCa\textsubscript{v}3) from
a fly head cDNA
library of adult heads including the brain. library.
The sequence of cloned cDNA was exactly matched
[Which one?] to an isoform predicted in
href{http://flybase.org/reports/FBgn0264386.html}{Flybase}. FlyBase (\href{http://}{http://flybase.org}).
To examine the electrophysiological properties of DmCa\textsubscript{v}3, we injected cRNAs made from the DmCa\textsubscript{v}3 cDNA template into \emph{Xenopus} oocytes.
From 4 days after cRNA injection, DmCa\textsubscript{v}3 was expressed as measured by robust inward currents in 10 mM Ba\textsuperscript{2+} as a charge carrier.
To directly compare the biophysical properties of DmCa\textsubscript{v}3 and mammalian T-type Ca\textsuperscript{2+} channel homolog under the same conditions, we expressed rat Ca\textsubscript{v}3.1 subunit of which biophysical properties were previously reported in the expression
system3. system\cite{9495342}.
Compared to Ca\textsubscript{v}3.1, DmCa\textsubscript{v}3 had a similar low-voltage threshold (around -60 mV) for activation, although the averaged value is slightly lower by 3 ~ 4 mV.
The current traces of DmCa\textsubscript{v}3 and Ca\textsubscript{v}3.1 were activated and then inactivated during serial step pulses from a holding potential of -90 mV, producing transient current kinetics with the inactivation kinetics of DmCa\textsubscript{v}3 currents being likely to be slightly slower than those of Ca\textsubscript{v}3.1 currents.
The activation and inactivation kinetics of currents through DmCa\textsubscript{v}3 was accelerated as the higher step pulses were applied, producing a criss-crossing pattern (Fig.
1A, left), 1a), a typical T-type Ca\textsuperscript{2+} channel kinetics.
Analysis of current-voltage (I-V) relationships showed that
V50,act V\textsubscript{50,act} for half-maximal activation and slope factor (k) of DmCa\textsubscript{v}3 channel are -43.32 $\pm$ 1.38 mV and 7.74 $\pm$ 1.33, while those of Ca\textsubscript{v}3.1 are -38.92 $\pm$ 0.99 and 6.35 $\pm$ 0.94 (Fig.
1A, right). 1a).
These results suggest that DmCa\textsubscript{v}3 can be activated more negative potential than Ca\textsubscript{v}3.1 by 4.4 mV.
Taken together, the biophysical properties of DmCa\textsubscript{v}3 including activation threshold of about -60 mV, formation of maximal current amplitude at -20 mV, transient current kinetics, a criss-crossing pattern by currents evoked by a voltage protocol for I-V are very similar to the hallmark properties of native T-type Ca\textsuperscript{2+} channels as well as cloned
channels3-6. channels\cite{9495342}.
The activation curves obtained from fitting chord conductance with a Boltzmann equation showed that the potentials (V50,act) for half-maximal activation of DmCa\textsubscript{v}3 and Ca\textsubscript{v}3.1 are -43.32 $\pm$ 1.58 and -38.92 $\pm$ 1.15 mV, respectively, indicating that DmCa\textsubscript{v}3 channel activated at 4.4 mV lower test potentials than Ca\textsubscript{v}3.1 (P < 0.05, Student's t-test, n=10 -- 14) (Fig. 1B).
In regard to window current typically designated by the portion overlapped in the steady-state inactivation and activation curves, the window region for DmCa\textsubscript{v}3 is significantly larger than that for Ca\textsubscript{v}3.1, implying that DmCa\textsubscript{v}3 is capable of persistently evoking higher channel activity over relevant voltage range than Ca\textsubscript{v}3.1.
...
Analysis of dose-response curves shows that the IC\textsubscript{50} values for DmCa\textsubscript{v}3 and Ca\textsubscript{v}3.1 are 5.12 and 276.5 $\mu$M, respectively (Fig. 1F, right).
These results indicate that the Ni\textsuperscript{2+} block of DmCa\textsubscript{v}3 currents is ~50 fold more sensitive than that of Ca\textsubscript{v}3.1 , suggesting that the nickel sensitive block of DmCa\textsubscript{v}3 channel resembles Ca\textsubscript{v}3.2 T-type channel (Table \ref{tab:1}).
