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\section*{Results}  \subsection*{DmCa\textsubscript{v}3 channel produces LVA currents in \emph{Xenopus} oocytes}  To determine biophysical properties of 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 from cDNA library of adult heads including the brain.  The sequence of cloned cDNA was exactly matched [Which one?] isoform predicted in href{http://flybase.org/reports/FBgn0264386.html}{Flybase}.  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.  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), a typical T-type Ca\textsuperscript{2+} channel kinetics.  Analysis of current-voltage (I-V) relationships showed that V50,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).  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.  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.  Voltage-dependent kinetics are known to different among three mammalian T-type calcium channels7.  Ca\textsubscript{v}3.1 and Ca\textsubscript{v}3.2 shows similar activation/inactivation kinetics whereas Ca\textsubscript{v}3.3 has much slower kinetics.  We compared the time constants of activation and inactivation by fitting the current traces with double exponential function.  Over the voltage ranges tested, DmCa\textsubscript{v}3 has slower current kinetics than Ca\textsubscript{v}3.1 (P< 0.01 or 0.001, Fig. 1C).  DmCa\textsubscript{v}3 has activation kinetics of ? ms and inactivation kinetics of ? ms at 10 mV potential, displaying about 2-fold slower kinetics than Ca\textsubscript{v}3.1 (Table 1).  Previous studies have shown that the current amplitude of Ca\textsubscript{v}3.1 in Ca\textsubscript{v}3 as a charge carrier is greater than in equi-molar Ba\textsuperscript{2+}, while the opposite is true for Ca\textsubscript{v}3.2 or Ca\textsubscript{v}3.3\cite{mcrory:2000aa,shcheglovitov:2007aa}.  Herein, we determined relative permeability (ICa/IBa) of Ca\textsubscript{v}3 and Ba\textsuperscript{2+} ions through DmCa\textsubscript{v}3 or Ca\textsubscript{v}3.1.  As reported, the ICa/IBa ratio of Ca\textsubscript{v}3.1 is 1.55 $\pm$ 0.03 (n=5), showing that the peak current amplitude of Ca\textsubscript{v}3.1 is greater in 10 mM Ca\textsubscript{v}3 than 10 mM Ba\textsuperscript{2+} (Fig. 1D and 1E, left).  On the contrary, the ICa/IBa of DmCa\textsubscript{v}3 channel is 0.68 $\pm$ 0.04, indicating that the current amplitude through DmCa\textsubscript{v}3 channels in 10 mM Ca\textsubscript{v}3 solution is smaller than in 10 mM Ba\textsuperscript{2+} solution (n=5) (Fig. 1D and 1E, left).  These findings suggest that the relative permeability (ICa/IBa) of DmCa\textsubscript{v}3 channel resembles the property of Ca\textsubscript{v}3.2 or Ca\textsubscript{v}3.3 rather than Ca\textsubscript{v}3.1 (Table 1).  We also compared relative permeability of the two divalent ions through DmCa\textsubscript{v}3 and Ca\textsubscript{v}3.1 using their maximal slope conductance ratios (GMaxCa/GMaxBa).  The GMaxCa/GMaxBa ratios for DmCa\textsubscript{v}3 and Ca\textsubscript{v}3.1 are 0.71 $\pm$ 0.10 and 1.43 $\pm$ 0.17, respectively (n=6, 4) (Fig. 1E, right), supporting that Ca\textsubscript{v}3 permeation through DmCa\textsubscript{v}3 channel are less than Ba\textsuperscript{2+} permeation.  Sensitivity to Ni\textsuperscript{2+}, a blocker of T-type Ca\textsuperscript{2+} channel, has been a criteria that is differential according to T-type channel isoforms10.  Among three subtypes, Ca\textsubscript{v}3.2 is more sensitive to Ni\textsuperscript{2+} than Ca\textsubscript{v}3.1 and Ca\textsubscript{v}3.2.  Application of low micro-molar concentrations of Ni\textsuperscript{2+} solutions inhibited DmCa\textsubscript{v}3 currents in a concentration dependent manner (Fig. 1F, left).  In contrast, Ca\textsubscript{v}3.1 currents required much higher concentrations of Ni\textsuperscript{2+} solutions to be blocked (Fig. 1F, left).  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}).