Kyunghwa Jeong edited Results_electrophys.tex  almost 9 years ago

Commit id: 6e21f29e1e2be7905eb92ed92127c8125e5ea357

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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 system\cite{9495342}.  Compared to Ca\textsubscript{v}3.1, DmCa\textsubscript{v}3 had a similar low-voltage threshold (around \textminus60 mV) for activation, although the averaged value is slightly lower by 3 \sim 3\sim  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 \textminus90 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), a typical T-type Ca\textsuperscript{2+} channel kinetics.  Analysis of current-voltage (I-V) relationships showed that V\textsubscript{50,act} for half-maximal activation and slope factor (k) of DmCa\textsubscript{v}3 channel are \textminus43.32 $\pm$ 1.38 mV and 7.74 $\pm$ 1.33, while those of Ca\textsubscript{v}3.1 are \textminus38.92 $\pm$ 0.99 and 6.35 $\pm$ 0.94 (Fig. 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 \textminus60 mV, formation of maximal current amplitude at \textminus20 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 channels\cite{9495342}. channels\cite{9495342, 6087159, 9670923, 10066244}.  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.