pbio/neubehav 550: biophysics of ca 2+ signaling week 4 (04/22/13) calcium transport and buffers
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PBio/NeuBehav 550: Biophysics of Ca 2+ signaling Week 4 (04/22/13) Calcium transport and buffers. Thoughts for today: Ca 2+ transporters shuffle Ca 2+ around the cell to regulate activity Ca 2+ switches bind and buffer Ca 2+ Buffers change function. Ca 2+ fluxes in an excitable cell. - PowerPoint PPT PresentationTRANSCRIPT
PBio/NeuBehav 550: Biophysics of Ca2+ signalingWeek 4 (04/22/13)
Calcium transport and buffers
Thoughts for today:
• Ca2+ transporters shuffle Ca2+ around the cell to regulate activity
• Ca2+ switches bind and buffer Ca2+
• Buffers change function
ER
SOC/CRAC channel
PM Ca2+ ATPase
Na+-Ca2+ exchanger
Plasma membrane
Ca2+
Na+
Ca2+
IP3R channel
Ca2+
Typical Ca2+ fluxes in a pituitary cell
Responses: Exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression
Ca2+ fluxes in an excitable cell
Inputs: hormones, synaptic inputs, cytokines, growth factors
GqPLC
AgonistR
IP3
DAG
ATP
ATP
Ca2+
MitoCa2+
Na+
LDCSG nucleus
VG Ca channels
SERCA pump
Ca2+
PIP2
Anterior pituitary control by portal peptide factors
Brainrostral
Hypothalamus
Anteriorpituitary
Posteriorpituitary
Blood
FSH/LH
GnR
H
GnRHOscill93
Time (s)
I K(C
a) (
pA)
GnRH
0 100 200 3000
2
0
100
(Tse & Hille, 1992)
GnRH makes Ca2+ and IK(Ca) oscillateC
a2+] i
(M
)
[Ca2+]
I K(Ca)
gonadotrope loaded by pipette with 50 M indo1
GnRH induces oscillatory exocytosis synchronous with Ca2+
(Tse & Hille, Science, 1992)Time (s)
0
40 nM GnRHdC
m/d
t fF
/s
0
2
0
10050
pituitary gonadotroph
Cm
(fF
)
0
600
Ca2
+ (M
)
150
calcium
exocytosis rate
membrane area
Ca2+
Ca2+ suffices. Other PLC products are not essential.
(Tse, Tse, Hille, Horstmann, Almers, Neuron, 1997)
gonadotrope with caged Ca in pipette
Time (s)0P
lasm
a m
embr
ane
area
cha
nge
(Cm
fF
)
600
0
1 2
400
200
Before flashCai = 100 nM
After flashCai = 50 M
UV flash release Cai
from DM nitrophen
growingmembrane area
Ca2+
GonadoCaFree
Time (s)
Ca2+ influx is not required
(Tse & Hille, 1992)
0 100 200 300 4000
0.8
0.4
0 Ca2+ (EGTA)
Ca2
+] i
(M
)hormone-activated gonadotrope with 50 M indo1
ERCa2+
Ca2+ oscillations need the SERCA pump
BHQ, a readily reversible blocker of SERCA pumps arrests Ca2+ oscillations at the cytoplasmic high-Ca2+ level. (Tse, Tse, Hille, PNAS, 1994)
Time (s)
0
2 nM GnRH
0.5
1.5
0
400200
pituitary gonadotrope
1 10 M BHQER
Ca2+
Ca2
+] i
(M
)
Dye loading in intracellular stores of gonadotrope
ER
Mag-Indo1 AM
ER
Mn2+
Mn2+
Preloading Unloading & quenching(Tse, Tse, Hille, PNAS, 1994)
Brightfield Epifluorescence)
0 s 30 s 60 s
Mag-Indo1 is a Ca reporter with a low Ca affinity (~35 uM)
60 s
GnRH releases Ca2+ from stores
Time (s)0
2 nM GnRHIK
(Ca
)
0
50
Pituitary gonadotroph patch clamped and loaded with Mag-indo-1 in ER com-partments. (Tse, Tse, Hille, PNAS, 1994)
sto
res
Ca2
+ (M
)
calcium depletes in stores
"cytoplasmic calcium"
stores calcium
cytoplasmic calcium
Time (s)
25
250 500
32
60
Steady state
ERCa2+
Some Ca goes missing!!
?
ER
SOC/CRAC channel
SERCA pump
PM Ca2+ ATPase
Na+-Ca2+ exchanger
Plasma membrane
Ca2+
Na+
Ca2+
IP3R channel
Ca2+
Typical Ca2+ fluxes in a pituitary cell
Responses: Exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression
Ca2+ fluxes in an excitable cell
Inputs: hormones, synaptic inputs, cytokines, growth factors
GqPLC
AgonistR
PIP2
IP3
DAG
ATP
ATP
Ca2+
Ca2+
MitoCa2+
Na+
LDCSG nucleus
VG Ca channels
ChromCCCP
Time (s)
Mitochondrial Ca2+ clearance dominates in chromaffin cells
0 60 120
Cyt
opla
smic
[C
a2+] i
(M
)
chromaffin cell loaded with indo1
(Herrington, Park, Babcock, Hille, 1996)
0
1
2
A 1-s depolarization loads cell with calcium. Clearance then begins.
control
CCCP
CCCP collapses proton motive force
Rate of fall is a measure of rate of Ca clearance from cytoplasm without mitochondrial uptake
ChromCCCP2
Time (s)
Mitochondria store Ca2+ for a while; CCCP lets it out
0 30 60 90 120
chromaffin cell loaded with indo1
(Herrington, Park, Babcock, Hille, 1996)
0
1
2
3
CCCP1
CCCP2
CCCP1
CCCP2
A 1-s depolarization loads cell with calcium. Clearance then begins.
CCCP stops uptake into mitochondria
Can we "see" Ca2+ in mitochondria?
Cyt
opla
smic
[C
a2+] i
(M
)
ChromDeconv96
Cationic rhod-2 accumulates in mitochondria
(Babcock, Herrington, Goodwin, Park, Hille, 1997)
chromaffin cell loaded with rhod-2
14 m
deconvolution microscopy
KCl
wash
ChromRhod2
Time (s)
Mitochondria pump Ca2+ back to cytoplasm
0 100 200 300
Ca2
+] c
yto
pl (
M)
(Babcock, Herrington, Goodwin, Park, Hille, 1997)
chromaffin cell loaded with rhod-2-AM and calcium green in pipette
Ca2
+] m
ito (M
)
1.0
0.5
0
0.2
0.4
0.6
mito. (rhod-2)
cyto. (CG)
Ca2+
Mito
ChromRhod2A
Rhod-2 is reporting mitochondrial Ca2+C
a2+] c
yto (M
)C
a2+] m
ito (M
)
(Babcock, Herrington, Goodwin, Park, Hille, 1997)
chromaffin cell loaded with rhod-2 AM and calcium green
200 soligomycin
0
0.5
1.0
0.1
0.5
calcium greencytoplasm
rhod2mitochondria
CCCP
ChromRates
free Ca2+]c (M)
Ca2+ transporter rates in chromaffin cells
0 0.5 1.0 1.5Tra
nspo
rt r
ate
(bou
nd +
fre
e) (M
/s)
(Herrington, Park, Babcock, Hille, 1996)
0
20
40
60
mitochondria
pmCa-ATPase
NCX
rest
These rates are calculated from slopes of [Ca] decay after a Ca load, multiplied by the cytoplasmic Ca binding ratio, to yield the actual moles crossing cell membranes.
Ca2+ clearance rates for three cell types
chromaffin cell
spermatozoon
0
40
80
0 1 2Ca2+]c (M)
total
SERCA
PMCA
NCX
0
2
mito
0 1.0Ca2+]c (M)
total
PMCA1
NCX
Ca2+]c (M)
Tra
nspo
rt r
ate
(M
/s)
0
20
40
60
mito
0 1.0
PMCA
NCX
3 clearance
Babcock/Herrington Chen/Koh Wennemuth
20
60
pancreatic beta cell
1950s: The Cambridge school
•Are ions free in the cytoplasm or are they bound?
H-K meth blue
After injection, spread of dye in one dimension (r) would follow the Einstein equation approximately ("bell-shaped" Gaussian distribution):
C(x,t) = Const. * (1/t) * exp –(r2/2Dt) SD = = sqrt(2Dt)
From this and dye data: find that D for a dye in axon is 1.5*10–6 cm2/s, compared to 4*10–6 cm2/s for dye in water. (Hodgkin & Keynes, 1956)
700 m
before 3 s 20 s 120 s 600 s
How fast do molecules diffuse in axons?
15 s
Generalization: In cells D is typically ½ of free-solution value so Gcyto= Gext / 2
Methylene blue is injected into a squid axon along its axis.
H-K Ca45 spread
45Ca2+ diffusion in axons
45Ca2+ is injected into a short stretch of axon and its longitudinal diffusion gives an effective diffusion constant C(x,t) = Const. * (1/t) * exp –(r2/2Dt)DCa in axon = ~0.4*10–6cm2/s compared to 6*10–6 cm2/s in water.
14 min
478 min
–4 –2 0 2 4 6distance r (mm)
Hodgkin &Keynes, 1957
axon
gam
ma
co
unt
s
Free particles diffuse at their normal free rate DCa, but the total population diffuses more slowly. The total population diffuses at a rate DCa/(1 + if the bound complex can't move, or, more generally: Dfree + mobileDbound,mobile
(1 + mobile + immobile)
DCa DCa DCa DCa DCa
Caf(1)
Cabound
= Caf(1)
immobile
Caf(2)
Cabound
= Caf(2)
immobile
Caf(3)
Cabound
= Caf(3)
immobile
Caf(4)
Cabound
= Caf(4)
immobile
Binding slows diffusion
is the "calcium binding ratio"
Clearance is slowed too
A family of Ca2+-sensitive switches and buffers
Calmodulin
Parvalbumin is present in GABAergic interneurons in the nervous system especially the reticular thalamus] and chandelier and basket cells in the cortex. In the cerebellum, PV is expressed in Purkinje cells and molecular layer interneurons.]
Most of the PV interneurons are fast-spiking. They are also thought to give rise to gamma waves recorded in EEG.....
Calbindin-D28k is present in the intestine, kidney. and a number of neuroendo-crine cells, particularly in the cerebellum. Cerebellar Pukinje cells.
Calretinin CR is in interneurons of granule cell layer
(Antisense cerebellar images from Allen Brain Atlas, http://www.brain-map.org/)
GonadoModel
exocytosis
K(Ca) Ca
GnRHR Gq
IP3R
Mitoch.
ER
Ca2+LH
FSH
LHFSH
LHFSH
PLC DAGPLC
IP3
Buffers of a pituitary gonadotrope?
GnRH
GonadoCaBookkeep
cytosol ER stores mitochondria
Approx. volume 1 0.1 0.06 free (M) 1 10 0.4 bound (M) 100 1000 1700 ratio bound/free() 100 100 4000
Estimating Ca2+ binding ratios
calmodulin chaperones proteins??? calretinin calreticulin PO4
calbindin calnexin phospholipid?parvalbumin BIPannexins calsequestrin
The calculations combine experiments with gonadotropes and chromaffin cells
Candidate buffers:
Interlude for discussing Augustine/Neher paper
Discussion of Neher/Augustine paper"Calcium gradients and buffers in bovine chromaffin
cells"Each figure will be fully described by a student--as if you are
teaching it to us for the first time. Further questions will come from the audience.
Purpose of paper Bertil
Fig. 4 Jerome Cattin
Fig. 5 Jacob Baudin
Fig. 6 Andrea McQuate
Fig. 7 Jesse Macadangdang
Fig. 8 Benjamin Drum
Fig. 9 Anastasiia Stratiievska
AN4Fig 4 Jerome Cattin 100 ms 300 ms 500 ms
1,000 ms after end 10,000 ms after end
Fig 5 Jacob Baudin
AN5
rest level
Diffusion into a sphere of radius r
(Crank, The Mathematics of Diffusion, Oxford, 1956)see also Carslaw & Jaeger, The Conduction of Heat in Solids, Oxford
x = 0, centerof sphere
x = r, edgeof sphere
Modeled times are given in multiples of the diffusional characteristic time: r2 / D For example, if a cell has radius r = 9 m and the free diffusion coefficient is 4 * 10–6 cm2/s as for small ions. Then r2/D is 20 ms, and for the red curve labeled 0.15: t = 0.15 r2/D = 0.15x20 ms corresponds to 3 ms.
distance from center of sphere
Crank in sphere
0.15
AN & Crank
Since Ca takes perhaps 50-100 ms instead of 3 ms to reach the 0.15 curve, it might be ~30 times less mobile than free Ca in this chromaffin cell experiment with EGTA & fura.
Rough guesstimate of Ca2+ diffusion rate
Fig. 6 Andrea McQuate
AN6
500 ms
rest level
Fura
Fig 7 Jesse Macadangdang
AN7
500 ms
250 ms
ICa
Fura
Fura
1400
1200
1000
800
600
400
200
0
1.21.00.80.60.40.20.0
Binding ratios depend on indo and Ca concentrations as well as endogenous buffer
Diff
eren
tial C
a bi
ndin
g ra
tio ()
Ca2+]i (M)
= 100+ (cindo/Kindo)/(1+Cai/Kindo)2
suppose endogenous = 100, then added indo-1 increases above 100
0 M
100
200
300 600 M
500 M
400 M of added indo-1
Indo Binding Ratio
Ca bound to indo = cindo/(1+Kindo/Cai)
Kindo ~ 200 nM
Fig 8 Benjamin Drum AN8
Inset
400 uM fura-2
back to 50 uM fura-2
= 190 s
Fig 9 Anastasiia Stratiievska
AN9
(seconds)
Fura-2 Ca binding ratio (B)
? ?
-89
7 s
de
cay
(s)
Conclusions :Binding = buffering & sensing
Buffering reduces Ca2+ changes,Slows Ca2+ changes,
Slows diffusion, Shortens local spikes of Ca2+