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+