deletions | additions
diff --git a/Methods_oocytes.tex b/Methods_oocytes.tex
index 35e04f7..cb1b90c 100644
--- a/Methods_oocytes.tex
+++ b/Methods_oocytes.tex
...
The GenBank accession number of the DmCa\textsubscript{v}3 is NP001096889.
Linearized cDNAs encoding rat Ca\textsubscript{v}3.1 or DmCa\textsubscript{v}3 were used as a templates for synthesis of capped cRNAs using T7 RNA polymerase (Ambion, Austin, TX, USA).
The concentrations of cRNAs were estimated based on optical density measured at 260 nm with a spectrophotometer.
Preparation of oocytes from female \emph{Xenopus laevis} frogs and injection of rat Ca\textsubscript{v}3.1 cRNAs or DmCa\textsubscript{v}3 cRNAs into oocytes were performed according to the previously reported methods
\cite{16377633}. \cite{kang:2006aa}.
diff --git a/Methods_sleep.tex b/Methods_sleep.tex
index f08187f..f329324 100644
--- a/Methods_sleep.tex
+++ b/Methods_sleep.tex
...
Sleep and locomotor behavior of flies were measured using Drosophila Activity Monitoring system (Trikinetics).
For sleep analysis, 3-4 day-old female flies were placed into 65 mm X 5mm glass tube individually with one end filled with 2\% agar/ 5\% sucrose food and the other end capped by cotton ball.
Activity was monitored as every bim break in 1 minute bin and 5 minutes and more inactivity were counted as sleep \cite{Shaw:2000ui}.
After one day of habituation in the incubator (25$\,^{\circ}\mathrm{C}$, 60\% humidity), sleep during two days of 12hr:12hr light-dark cycle and following two days of dark period was analysed using Counting Macro
\cite{21041393}. \cite{pfeiffenberger:2010ab}.
For circadian locomotor analysis, 1-3 day-old male flies were used. Activity was measured every 30 min bin and analysed using ClockLab analysis software (Actimetrics) and Counting
Macro\cite{21041392}. Macro\cite{pfeiffenberger:2010aa}. Significance level of the $\chi$\textsuperscript{2} periodogram was set to $\alpha$ = 0.05.
Flies with a power of significance (P-S) $\geq$10 were considered as rhythmic.
diff --git a/Results_clock.tex b/Results_clock.tex
index 6878657..94df666 100644
--- a/Results_clock.tex
+++ b/Results_clock.tex
...
\subsection*{DmCa\textsubscript{v}3 mutant does not affect core clock system}
Sleep is regulated by circadian system and some clock mutants show altered sleep
phenotype\cite{12568241, 19038223}. phenotype\cite{hendricks:2003aa, parisky:2008aa}.
We examined whether this increased sleep phenotype is due to altered circadian clock.
Activity of \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} was monitored for seven days of continuous darkness.
\emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} flies showed rhythmic activity until the 7th day of continuous dark period although they have increased period length and reduced rhythmicity compared to \emph{w\textsuperscript{1118}} (Fig. \ref{fig:4}a). The reduced percentage of rhythmic flies (90.7\%) and power of rhythmicity (24.0 $\pm$ 3.4) in \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} are probably due to the increased sleep at the transition of subjective day and subjective night.
diff --git a/Results_electrophys.tex b/Results_electrophys.tex
index 2024f32..4af92e1 100644
--- a/Results_electrophys.tex
+++ b/Results_electrophys.tex
...
At the test potentials from \textminus50 mV to +20 mV, DmCa\textsubscript{v}3 has slower current kinetics than Ca\textsubscript{v}3.1 (P $<$ 0.01 or 0.001, Fig. \ref{fig:1}c).
For example, the activation and inactivation time constants of DmCa\textsubscript{v}3 current at \textminus20 mV test potential are 2.2 $\pm$ 0.2 ms and 23.4 $\pm$ 1.4 ms respectively, showing about 2-fold slower activation and inactivation kinetics than those of rat Ca\textsubscript{v}3.1 current (Table \ref{tab:1}).
One of defining properties of T-type calcium channels is that they deactivate slowly compared to the HVA calcium channels that have much faster deactivation
kinetics\cite{PerezReyes:1998gn,10066244,2419479}. kinetics\cite{PerezReyes:1998gn,10066244,matteson:1986aa}.
For characterizing the deactivation kinetics of DmCa\textsubscript{v}3, we transiently expressed DmCa\textsubscript{v}3 cDNA in HEK-293 cells and recorded tail currents using whole cell patch clamping.
The tail currents of DmCa\textsubscript{v}3 appeared to be slowly decayed in a voltage dependent manner (Fig. \ref{fig:1}d).
The deactivation time constants from fitting the tail currents are most similar to mammalian Ca\textsubscript{v}3.3 among the three Ca\textsubscript{v}3 isoforms previously reported (Table \ref{tab:1}).
diff --git a/bibliography/references.bib b/bibliography/references.bib
index 46db900..a0dca3e 100644
--- a/bibliography/references.bib
+++ b/bibliography/references.bib
...
%% http://bibdesk.sourceforge.net/
%% Created for Walton Jones at
2015-07-18 21:20:08 2015-07-20 10:48:13 +0900
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Bdsk-Url-1 = {http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=15601764&retmode=ref&cmd=prlinks},
Bdsk-Url-2 = {http://dx.doi.org/10.1073/pnas.0408089101}}
@article{2419479, @article{matteson:1986aa,
Author = {Matteson, DR and Armstrong, CM},
Authors = {Matteson, DR and Armstrong, CM},
Date = {1986 Jan},
Date-Modified = {2015-07-20 01:29:07 +0000},
Doi = {10.1085/jgp.87.1.161},
Journal = {J Gen Physiol},
Month = {Jan},
Pages = {161-82},
...
Source = {J Gen Physiol},
Title = {{Properties of two types of calcium channels in clonal pituitary cells.}},
Volume = {87},
Year =
{1986}} {1986},
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@article{15016809, @article{park:2004aa,
Author = {Park, JY and Kang, HW and Jeong, SW and Lee, JH},
Authors = {Park, JY and Kang, HW and Jeong, SW and Lee, JH},
Date = {2004 May 21},
Date-Modified = {2015-07-20 01:45:27 +0000},
Doi = {10.1074/jbc.M400684200},
Journal = {J Biol Chem},
Month = {May},
Pages = {21707-13},
...
Source = {J Biol Chem},
Title = {{Multiple structural elements contribute to the slow kinetics of the Cav3.3 T-type channel.}},
Volume = {279},
Year =
{2004}} {2004},
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@article{23970551, @article{demers-giroux:2013aa,
Author = {Demers-Giroux, PO and Bourdin, B and Sauv{\'e}, R and Parent, L},
Authors = {Demers-Giroux, PO and Bourdin, B and Sauv{\'e}, R and Parent, L},
Date = {2013 Oct 11},
Date-Modified = {2015-07-20 01:30:34 +0000},
Doi = {10.1074/jbc.M113.500975},
Journal = {J Biol Chem},
Month = {Oct},
Pages = {29281-93},
...
Source = {J Biol Chem},
Title = {{Cooperative activation of the T-type CaV3.2 channel: interaction between Domains II and III.}},
Volume = {288},
Year =
{2013}} {2013},
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@article{11927664, @article{chemin:2002aa,
Author = {Chemin, J and Monteil, A and Perez-Reyes, E and Bourinet, E and Nargeot, J and Lory, P},
Authors = {Chemin, J and Monteil, A and Perez-Reyes, E and Bourinet, E and Nargeot, J and Lory, P},
Date = {2002 Apr 1},
Date-Modified = {2015-07-20 01:33:38 +0000},
Doi = {10.1113/jphysiol.2001.013269},
Journal = {J Physiol},
Month = {Apr},
Pages = {3-14},
...
Source = {J Physiol},
Title = {{Specific contribution of human T-type calcium channel isotypes (alpha(1G), alpha(1H) and alpha(1I)) to neuronal excitability.}},
Volume = {540},
Year =
{2002}} {2002},
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@article{16377633, @article{kang:2006aa,
Author = {Kang, HW and Park, JY and Jeong, SW and Kim, JA and Moon, HJ and Perez-Reyes, E and Lee, JH},
Authors = {Kang, HW and Park, JY and Jeong, SW and Kim, JA and Moon, HJ and Perez-Reyes, E and Lee, JH},
Date = {2006 Feb 24},
Date-Modified = {2015-07-20 01:46:57 +0000},
Doi = {10.1074/jbc.M510197200},
Journal = {J Biol Chem},
Month = {Feb},
Pages = {4823-30},
...
Source = {J Biol Chem},
Title = {{A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels.}},
Volume = {281},
Year =
{2006}} {2006},
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@article{21041393, @article{pfeiffenberger:2010ab,
Author = {Pfeiffenberger, C and Lear, BC and Keegan, KP and Allada, R},
Authors = {Pfeiffenberger, C and Lear, BC and Keegan, KP and Allada, R},
Date = {2010 Nov 1},
Date-Modified = {2015-07-20 01:39:55 +0000},
Doi = {10.1101/pdb.prot5520},
Journal = {Cold Spring Harb Protoc},
Month = {Nov},
Pages = {pdb.prot5520},
...
Source = {Cold Spring Harb Protoc},
Title = {{Processing sleep data created with the Drosophila Activity Monitoring (DAM) System.}},
Volume = {2010},
Year =
{2010}} {2010},
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@article{21041392, @article{pfeiffenberger:2010aa,
Author = {Pfeiffenberger, C and Lear, BC and Keegan, KP and Allada, R},
Authors = {Pfeiffenberger, C and Lear, BC and Keegan, KP and Allada, R},
Date = {2010 Nov 1},
Date-Modified = {2015-07-20 01:38:25 +0000},
Doi = {10.1101/pdb.prot5519},
Journal = {Cold Spring Harb Protoc},
Month = {Nov},
Pages = {pdb.prot5519},
...
Source = {Cold Spring Harb Protoc},
Title = {{Processing circadian data collected from the Drosophila Activity Monitoring (DAM) System.}},
Volume = {2010},
Year =
{2010}} {2010},
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@article{12568241, @article{hendricks:2003aa,
Author = {Hendricks, JC and Lu, S and Kume, K and Yin, JC and Yang, Z and Sehgal, A},
Authors = {Hendricks, JC and Lu, S and Kume, K and Yin, JC and Yang, Z and Sehgal, A},
Date = {2003 Feb},
Date-Modified = {2015-07-20 01:36:11 +0000},
Doi = {10.1177/0748730402239673},
Journal = {J Biol Rhythms},
Month = {Feb},
Pages = {12-25},
...
Source = {J Biol Rhythms},
Title = {{Gender dimorphism in the role of cycle (BMAL1) in rest, rest regulation, and longevity in Drosophila melanogaster.}},
Volume = {18},
Year =
{2003}} {2003},
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@article{19038223, @article{parisky:2008aa,
Author = {Parisky, KM and Agosto, J and Pulver, SR and Shang, Y and Kuklin, E and Hodge, JJ and Kang, K and Liu, X and Garrity, PA and Rosbash, M and Griffith, LC},
Authors = {Parisky, KM and Agosto, J and Pulver, SR and Shang, Y and Kuklin, E and Hodge, JJ and Kang, K and Liu, X and Garrity, PA and Rosbash, M and Griffith, LC},
Date = {2008 Nov 26},
Date-Modified = {2015-07-20 01:43:42 +0000},
Doi = {10.1016/j.neuron.2008.10.042},
Journal = {Neuron},
Month = {Nov},
Pages = {672-82},
...
Source = {Neuron},
Title = {{PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit.}},
Volume = {60},
Year =
{2008}} {2008},
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@article{23849427, @article{park:2013aa,
Author = {Park, HJ and Park, SJ and Ahn, EJ and Lee, SY and Seo, H and Lee, JH},
Authors = {Park, HJ and Park, SJ and Ahn, EJ and Lee, SY and Seo, H and Lee, JH},
Date = {2013 Sep},
Date-Modified = {2015-07-20 01:26:13 +0000},
Doi = {10.1016/j.ceca.2013.06.006},
Journal = {Cell Calcium},
Month = {Sep},
Pages = {226-35},
...
Source = {Cell Calcium},
Title = {{Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Ca(v)3.2 T-type channel.}},
Volume = {54},
Year =
{2013}} {2013},
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diff --git a/discussion.tex b/discussion.tex
index 1b3a115..7958a59 100644
--- a/discussion.tex
+++ b/discussion.tex
...
Biophysical properties of the cloned channel are consistent with previous studies showing that DmCa\textsubscript{v}3 mediates low-voltage-activated (LVA) currents both in central and peripheral nervous system\cite{Ryglewski:2012jk, Iniguez:2013ib}.
Analysis of the fly genome suggests that DmCa\textsubscript{v}3 is unique T-type channel isoform in the fly, which is different from existence of the three isoforms (Ca\textsubscript{v}3.1-3.3) in mammals.
In current kinetics of view, DmCa\textsubscript{v}3 is similar to mammalian Ca\textsubscript{v}3.1 and Ca\textsubscript{v}3.2 rather than mammalian Ca\textsubscript{v}3.3 with much slower current kinetics.
In terms of relative permeability and nickel inhibition sensitivity, the preferential permeation of Ba\textsuperscript{2+} over Ca\textsuperscript{2+} and nickel sensitive inhibition of DmCa\textsubscript{v}3 suggest that the fly T-type channel resembles to mammalian
Ca\textsubscript{v}3.2\cite{16377633, 23849427}. Ca\textsubscript{v}3.2\cite{kang:2006aa, park:2013aa}.
Broad expression patterns of DmCa\textsubscript{v}3 in various region of adult brain are reminiscent of the mammalian T-type Ca\textsuperscript{2+} channels widely distributed throughout the brain\cite{talley:1999aa}.
Three subtypes of mammalian T-type Ca\textsuperscript{2+} channels are express in largely complementary manner with distinct biophysical properties, which confers the functional diversity.
diff --git a/table.tex b/table.tex
index a1a6782..39b26e3 100644
--- a/table.tex
+++ b/table.tex
...
\multicolumn{1}{l}{{\bf Inactivation}} & & & & & \\ \midrule \addlinespace
V\textsubscript{50} (mV) & \textminus58.0 $\pm$ 0.7 (5) & \textminus61.3 $\pm$ 0.7 (15) & \textminus64.9 $\pm$ 0.7\cite{park:2006aa} & \textminus62.0 $\pm$ 0.9\cite{park:2006aa} & {\bf \textminus55.6 $\pm$ 0.9\cite{park:2006aa}} \\ \addlinespace \addlinespace
\multicolumn{1}{l}{{\bf Current kinetics (\textminus20 mV)}} & & & & & \\ \midrule \addlinespace
$\tau$\textsubscript{act} (ms) & 2.2 $\pm$ 0.2 (10) & 1.1 $\pm$ 0.2 (5) & {\bf 1.4 $\pm$
0.1\cite{15016809}} 0.1\cite{park:2004aa}} & 3.2 $\pm$
0.1\cite{23970551} 0.1\cite{demers-giroux:2013aa} & 22.8 $\pm$
1.5\cite{15016809} 1.5\cite{park:2004aa} \\ \addlinespace
$\tau$\textsubscript{inact} (ms) & 23.4 $\pm$ 1.4 (6) & 9.7 $\pm$ 0.9 (5) & 7.2 $\pm$
0.4\cite{15016809} 0.4\cite{park:2004aa} & {\bf 11.6 $\pm$
0.2\cite{23970551}} 0.2\cite{demers-giroux:2013aa}} & 97.0 $\pm$
3.8\cite{15016809} 3.8\cite{park:2004aa} \\ \addlinespace \addlinespace
\multicolumn{1}{l}{{\bf Deactivation kinetics (\textminus100 mV)}} & & & & & \\ \midrule \addlinespace
$\tau$\textsubscript{deact} (ms) & 0.93 $\pm$ 0.14 (6)\tnote{a} & NA & 2.6 $\pm$
0.2\tnote{a}\textsuperscript{,}\tnote{b}\cite{11927664} 0.2\tnote{a}\textsuperscript{,}\tnote{b}\cite{chemin:2002aa} & 3.6 $\pm$
0.4\tnote{a}\textsuperscript{,}\tnote{b}\cite{11927664} 0.4\tnote{a}\textsuperscript{,}\tnote{b}\cite{chemin:2002aa} & {\bf 1.12 $\pm$
0.1\tnote{a}\textsuperscript{,}\tnote{b}\cite{11927664}} 0.1\tnote{a}\textsuperscript{,}\tnote{b}\cite{chemin:2002aa}} \\ \addlinespace
\multicolumn{1}{l}{{\bf Conductance}} & & & & & \\ \midrule \addlinespace
I\textsubscript{Ca}/I\textsubscript{Ba} & 0.7 (6) & 1.6 (3) & 1.5\cite{shcheglovitov:2007aa} & 0.8\cite{shcheglovitov:2007aa} & {\bf 0.7\cite{shcheglovitov:2007aa}} \\ \addlinespace \addlinespace
\multicolumn{1}{l}{{\bf Pharmacology}} & & & & & \\ \midrule \addlinespace
