Post on 10-Feb-2020
Ca2+ and cell function
2001 Sinauer Associates, Inc.
2002 Alberts et al.
2004 Sinauer Associates, Inc.
2X10-3M (2mM)
10-7M(100nM)
10-4M(100 M)
Ca2+
Ca2+
Ca2+
http://sites.sinauer.com/neuroscience5e/animations07.02.html
Cells maintain a very low concentration of free Ca2+
in the cytosol
2002 Alberts et al.
Ca2+ Ca2+
Ca2+
Rhod-2Fura-2
Electroporation Calcium Green
0 20 40 60 80-0.2
0
0.2
0.4
0.6
0.8
1
Time (s)
DF/F0
#0#1#2
80400Time (s)
0
0.2
-60
-40
-20
0
20
40
60
Inverse e PPericamCa2+
Ca2+
Ca2+
LHβ /FSHβ
2kbInverse Pericam
PolyAsignal
LHβ-IP FSHβ-IP
IP
LH FSH Ca2+
LHβ-IP
FSHβ-IP
50μm
50μm
[Ca2+]i GnRH
LH FSH
[Ca2+]ii
CCD
Vh=-50mV
Exocytosis of GtH; blocked by EGTA/BAPTA in pipette
2001 Sinauer Associates, Inc.
Na+ K+
(mV
)
(mV
)
(mV
)
Na+
K+
Na+
(nA
)N
a+
(nA
)
K+
(nA
)K
+
(mA
/cm
2 )
Na+ K+
2004 Sinauer Associates, Inc.
Voltage-dependent Ca2+ channels
CsCl, TEA, EGTATTX 5mM Ca2+ A TTX 115mM Ba2+
B) -80mV -30mVC) -30mV
-80mV HVA, LVA
-30mV LVA
EH(-80)=HVA+LVAEH(-30)=HVA
HVALVA
HVALVA
-30mV LVAHVA
2001 Sinauer Associates, Inc.
EH = -30mV
EC = -20mV
LVA(EH = -80mV)
LVA LVA
LVA(EH = -80mV→EC = -20mV)
EH
EH
EC
EC
Ca2+
tiny conductancetransient current
large conductancelong-lasting current
2001 Sinauer Associates, Inc.
HVA HVA LVA
Tsien L P/Q N R T
α 1S 1C 1D 1F 1A 1B 1E 1G 1H 1I
CaV1.1CaV1.2CaV1.3CaV1.4CaV2.1CaV2.2CaV2.3CaV3.1CaV3.2CaV3.3
-30mV -20mV -70mV
( >500ms) ( 50~80ms) ( 20~50ms)
25pS 13pS 8pS
Cd2+>Ni2+ Cd2+>Ni2+ Ni2+>Cd2+
-CTX GVIA
-agatoxin
DHP
Ca2+
Hodgkin, A. & Huxley, A.1963
(Eccles, J.C. )
(stellate ganglion)
2004 Sinauer Associates, Inc.
Ca2+
(mV)
CaA/cm2
(mV)
2004 Sinauer Associates, Inc.
BAPTABAPTA
epsp EGTA
Ca2+, Ca2+ , Ca2+
Ca2+
Ca2+
EPSP
Ca2+
Ca2+
Ca2+
INa=gNa (Vm-ENa)
ICa=gCa (Vm-ECa)
Vm-ECa Tail current
gCa
INa IK
“Tail current”
-18mV+60mV
1ms 0.2ms
Ca2+ currents
Voltage clamp of presynaptic terminal
I2 vs V2
I1 vs V1
+TTX, TEA
+TTX, TEA
Ca2+
Ca2+
1 2
1:Ca2+
2:Ca2+
1+2=
2001 Sinauer Associates, Inc.
gNa
gCa
-18mV +60mV
IK=gK (Vm-EK)
Vm=EK
Ca2+
off Ca2+ “tail current”
Channels and transporters for Ca2+
ThapsigarginX
IP3R
2001 Sinauer Associates, Inc.
Ca2+ as a second messenger
triad
RyR
Phospholipase C- PI(4,5)P2
2002 Alberts et al.
IP3R RyR
GnRHCa2+
Ca2+
IP3 GnRH
Thapsigargin
Exocytosis of GtH; blocked by EGTA/BAPTA in pipette
Ca2+
: 10-7M[Ca2+]
Ca2+
Ca2+ Ca2+
10-7M~ 10-5M[Ca2+]
Thapsigargin
Thapsigargin
CRAC
2001 Sinauer Associates, Inc.
(A)
Pituitary gland and Pituitary gland and neurosecretory cells
SONPVN
Hypophysiotropic hormones
Pituitary
neurosecretory y cells (neuroendocrine)
Hypophysiotropic hormone-producing neurons
Hypothalamus
Pituitary
Hypophysiotropic system (Brain-Pituitary interface)
Gonadotropic hormones
(Posterior pituitary hormones)
Gonadotropin-releasing hormone (GnRH) system
1 2 3 4 5 6 7 8 9 10mGnRH pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
GnRH neurons
••
Protein and peptide hormones
Terminal Nerve GnRH System (sGnRH)
Midbrain GnRH System (cGnRH-II)
Hypophysiotrophic Preoptic GnRH System (sbGnRH)
Tel
TeO
ON
OB
Med
Pit
Cb
GnRH
Tel
Neuroendocrine
Neuromodulatory
Neuromodulatory
Gonadotropin-releasing hormone
pGlu-His -Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2
pGlu-His -Trp-Ser-Hisr -Gly-Trp-Tyr-Pro-Gly-NH2c -II
pGlu-His -Trp-Ser-Tyr-Gly-Leu-Ser-Pro-Gly-NH2seabream
GnRH3
GnRH2
GnRH1
Three paralogous genes for GnRH--Synteny analysisisis-
Okubo & Nagahama, 2007
Ancestral vertebrates
Ancestral tetrapods
Medaka
Dwarf Gourami
Dwarf gourami
Ancestral teleosts
Medaka
Dwarf Gourami
Hypophysiotropic hormone-producing neurons
Hypothalamus
Pituitary
Hypophysiotropic system (Brain-Pituitary interface)
Gonadotropic hormones
(Posterior pituitary hormones)
Gonadotropin-releasing hormone (GnRH) system
1 2 3 4 5 6 7 8 9 10mGnRH pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
GnRH neurons
1987 Kodansha Ltd.
Luteinizing hormone (LH)Follicle-stimulating hormone (FSH)Thyroid-stimulating hormone (TSH)
Growth hormone (GH)Prolactin (PRL)
Adrenocorticotropic hormone (ACTH)Melanocyte-stimulating hormone (MSH)
OxytocinVasopressin
Basophils (Glycoproteins)α
β
Acidophils
Chromatophobes
Pro-opiomelanocortin (POMC)
Protein and peptide hormones
Propressophysin Prooxyphysin
Neurophysin:
PC: proteolytic enzymesCLIP: corticotropin-like intermediate lobe peptide
LPH:
β-END: β
2000 Sinauer Associates, Inc.
1987 Kodansha Ltd.
1987 Kodansha Ltd.
Weltzien et al., 2004
FSH
LH
IP
LH FSH Ca2+
LHβ-IP
FSHβ-IP
50μm
50μm
[Ca2+]i GnRH
LH FSH
[Ca2+]ii
CCD
Pituitary
Pituitary
GFPFP-P-labelled GFFPP belled alaGnRH1 neurons
Brain
gnrh1:EGFP; lhb:IP
Inverse e Pericam
Ca2+
Modified from Karigo, et al., 2014
Caa2+++ imaging using Double transgenic medakaka: Cagnrh1
aging usimaih1h1:EGFP
g usPP;
ing DsusPP;;; lhb
Doung Dhbhb:IP
Na1
Na3
2
Na K4
55
2004 Sinauer Associates, Inc.
BioClassics5. /
Antidromic activation 1.2.
3.
VP
OX
A,B PVN
1,2 3,4
TNN-N-GnRHHTNNN-GGGnRHG HNN GGPacemaker potential)l)l)
Umatani et al., 2013 Kawai et al., 2013
Facilitatory modulation by GnRH3 peptide (Abe and Oka, 2000)
Saito et al., 2009
Inhibitory modulation by NPFF & RFRP
2 Steroid hormones
Aromatase