PBio/NeuBehav 550: Biophysics of Ca 2+ signaling Week 4 (04/22/13) Calcium transport and buffers

1. 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

2. Ca2+ fluxes in an excitable cell Typical Ca2+ fluxes in a pituitary cell Inputs: hormones, synaptic inputs, cytokines, growth factors PIP2 VG Ca channels Agonist DAG R PLC Na+-Ca2+ exchanger Gq IP3 Ca2+ Ca2+ Ca2+ IP3R channel SERCA pump Na+ ATP nucleus LDCSG Plasma membrane ER Ca2+ Ca2+ ATP PM Ca2+ ATPase Mito Ca2+ Na+ SOC/CRAC channel Responses: Exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression

3. Anterior pituitary control by portal peptide factors Hypothalamus Brain rostral Blood GnRH Posterior pituitary Anterior pituitary FSH/LH

4. GnRHOscill93 GnRH makes Ca2+ and IK(Ca) oscillate gonadotrope loaded by pipette with 50 M indo1 GnRH I K(Ca) 100 IK(Ca) (pA) 0 2 [Ca2+] [Ca2+]i (mM) 0 0 100 200 300 Time (s) (Tse & Hille, 1992)

5. GnRH induces oscillatory exocytosis synchronous with Ca2+ 40 nM GnRH pituitary gonadotroph 2 calcium Ca2+ (M) 0 Ca2+ 600  Cm (fF) membrane area 0 150 exocytosis rate dCm/dt fF/s 0 0 50 100 Time (s) (Tse & Hille, Science, 1992)

6. Ca2+ suffices. Other PLC products are not essential. gonadotrope with caged Ca in pipette UV flash release Cai from DM nitrophen 600 Before flash Cai = 100 nM After flash Cai = 50 M growing membrane area 400 Ca2+ Plasma membrane area change (Cm fF) 200 0 0 1 2 Time (s) (Tse, Tse, Hille, Horstmann, Almers, Neuron, 1997)

7. GonadoCaFree Ca2+ influx is not required hormone-activated gonadotrope with 50 M indo1 0.8 0 Ca2+ (EGTA) Ca2+ ER 0.4 [Ca2+]i (mM) 0 0 100 200 300 400 Time (s) (Tse & Hille, 1992)

8. Ca2+ oscillations need the SERCA pump pituitary gonadotrope 2 nM GnRH 1.5 Ca2+ ER 10 M BHQ 1 [Ca2+]i (mM) 0.5 0 0 200 400 Time (s) BHQ, a readily reversible blocker of SERCA pumps arrests Ca2+ oscillations at the cytoplasmic high-Ca2+ level. (Tse, Tse, Hille, PNAS, 1994)

9. Dye loading in intracellular stores of gonadotrope 0 s 30 s 60 s Epifluorescence) Brightfield Mag-Indo1 AM Mn2+ 60 s Mn2+ ER ER Mag-Indo1 is a Ca reporter with a low Ca affinity (~35 uM) Unloading & quenching Preloading (Tse, Tse, Hille, PNAS, 1994)

10. GnRH releases Ca2+ from stores ER Pituitary gonadotroph patch clamped and loaded with Mag-indo-1 in ER com-partments. (Tse, Tse, Hille, PNAS, 1994) 60 calcium depletes in stores stores Ca2+ (M) 2 nM GnRH 32 "cytoplasmic calcium" IK(Ca) 0 250 500 Time (s) Steady state stores calcium cytoplasmic calcium Ca2+ ? 0 25 50 Some Ca goes missing!! Time (s)

11. Ca2+ fluxes in an excitable cell Typical Ca2+ fluxes in a pituitary cell Inputs: hormones, synaptic inputs, cytokines, growth factors PIP2 VG Ca channels Agonist DAG R PLC Na+-Ca2+ exchanger Gq IP3 Ca2+ Ca2+ Ca2+ IP3R channel SERCA pump Na+ ATP nucleus LDCSG Plasma membrane ER Ca2+ ATP Ca2+ Mito PM Ca2+ ATPase SOC/CRAC channel Ca2+ Na+ Responses: Exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression

12. ChromCCCP Rate of fall is a measure of rate of Ca clearance from cytoplasm without mitochondrial uptake Mitochondrial Ca2+ clearance dominates in chromaffin cells chromaffin cell loaded with indo1 CCCP collapses proton motive force 2 CCCP Cytoplasmic [Ca2+]i (mM) 1 control 0 0 60 120 Time (s) A 1-s depolarization loads cell with calcium. Clearance then begins. (Herrington, Park, Babcock, Hille, 1996)

13. ChromCCCP2 Mitochondria store Ca2+ for a while; CCCP lets it out 3 chromaffin cell loaded with indo1 CCCP stops uptake into mitochondria 2 CCCP1 Cytoplasmic [Ca2+]i (mM) Can we "see" Ca2+ in mitochondria? 1 CCCP2 CCCP1 0 CCCP2 0 30 60 90 120 Time (s) A 1-s depolarization loads cell with calcium. Clearance then begins. (Herrington, Park, Babcock, Hille, 1996)

14. ChromDeconv96 Cationic rhod-2 accumulates in mitochondria chromaffin cell loaded with rhod-2 KCl wash 14 mm deconvolution microscopy (Babcock, Herrington, Goodwin, Park, Hille, 1997)

15. ChromRhod2 Mitochondria pump Ca2+ back to cytoplasm chromaffin cell loaded with rhod-2-AM and calcium green in pipette 1.0 0.6 Ca2+ Mito 0.4 mito. (rhod-2) [Ca2+]mito (mM) [Ca2+]cytopl (mM) 0.5 cyto. (CG) 0.2 0 0 100 200 300 Time (s) (Babcock, Herrington, Goodwin, Park, Hille, 1997)

16. ChromRhod2A Rhod-2 is reporting mitochondrial Ca2+ chromaffin cell loaded with rhod-2 AM and calcium green rhod2 mitochondria 0.5 [Ca2+]mito (mM) CCCP 0.1 1.0 calcium green cytoplasm 0.5 [Ca2+]cyto (mM) 200 s 0 oligomycin (Babcock, Herrington, Goodwin, Park, Hille, 1997)

17. ChromRates Ca2+ transporter rates in chromaffin cells 60 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. mitochondria 40 Transport rate (bound + free) (mM/s) 20 rest pmCa-ATPase NCX 0 0 0.5 1.0 1.5 free [Ca2+]c (mM) (Herrington, Park, Babcock, Hille, 1996)

18. 3 clearance Ca2+ clearance rates for three cell types pancreatic beta cell spermatozoon chromaffin cell 80 total 60 60 mito SERCA 40 40 2 Transport rate (mM/s) total PMCA NCX 1 20 20 NCX PMCA PMCA mito NCX 0 0 0 0 1.0 0 1 2 0 1.0 [Ca2+]c (mM) [Ca2+]c (mM) [Ca2+]c (mM) Babcock/Herrington Chen/Koh Wennemuth

19. 1950s: The Cambridge school • Are ions free in the cytoplasm or are they bound?

20. H-K meth blue How fast do molecules diffuse in axons? before 3 s 20 s 120 s 600 s 700 mm Methylene blue is injected into a squid axon along its axis. 15 s 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) Generalization: In cells D is typically ½ of free-solution value so Gcyto= Gext / 2

21. H-K Ca45 spread 45Ca2+ diffusion in axons Hodgkin & Keynes, 1957 14 min axon 478 min gamma counts –4 –2 0 2 4 6 distance r (mm) 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.

22. Difffsion w. binding Clearance is slowed too Binding slows diffusion DCa DCa DCa DCa DCa Caf(1) k Cabound = k Caf(1) immobile Caf(2) k Cabound = k Caf(2) immobile Caf(3) k Cabound = k Caf(3) immobile Caf(4) k Cabound = k Caf(4) immobile k is the "calcium binding ratio" 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 + k), if the bound complex can't move, or, more generally: Dfree + kmobile * Dbound,mobile (1 + kmobile + kimmobile)

23. 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-D28kis 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/)

24. GonadoModel Buffers of a pituitary gonadotrope? GnRH GnRHR Gq PLC DAG PLC IP3 ER IP3R LH FSH Ca2+ LH FSH Mitoch. LH FSH Ca K(Ca) exocytosis

25. GonadoCaBookkeep Estimating Ca2+ binding ratios cytosol ER stores mitochondria Approx. volume 1 0.1 0.06 D free (mM) 1 10 0.4 D bound (mM) 100 1000 1700 ratio bound/free(k) 100 100 4000 calmodulin chaperones proteins??? calretinin calreticulin PO4 calbindin calnexin phospholipid? parvalbumin BIP annexins calsequestrin Candidate buffers: The calculations combine experiments with gonadotropes and chromaffin cells

26. 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

27. Fig 4 Jerome Cattin AN4 100 ms 300 ms 500 ms 1,000 ms after end 10,000 ms after end

28. AN5 Fig 5 Jacob Baudin rest level

29. Diffusion into a sphere of radius r Crank in sphere x = 0, center of sphere x = r, edge of sphere Modeled times are given in multiples of the diffusional characteristic time: r2 / D For example, if a cell has radius r = 9 mm 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. 0.15 distance from center of sphere (Crank, The Mathematics of Diffusion, Oxford, 1956) see also Carslaw & Jaeger, The Conduction of Heat in Solids, Oxford

30. AN & Crank Rough guesstimate of Ca2+ diffusion rate 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.

31. AN6 Fig. 6 Andrea McQuate 500 ms Fura rest level

32. Fig 7 Jesse Macadangdang AN7 Fura 250 ms ICa Fura 500 ms

33. Indo Binding Ratio Binding ratios depend on indo and Ca concentrations as well as endogenous buffer Ca bound to indo = cindo/(1+Kindo/Cai) suppose endogenous k = 100, then added indo-1 increases k above 100 k = 100+ (cindo/Kindo)/(1+Cai/Kindo)2 600 mM 300 Kindo ~ 200 nM Differential Ca binding ratio (k) 500 mM 200 400 mM of added indo-1 100 0 mM [Ca2+]i (mM)

34. AN8 Fig 8 Benjamin Drum 400 uM fura-2 t = 190 s Inset back to 50 uM fura-2

35. AN9 ? ? Fig 9 Anastasiia Stratiievska (seconds)  decay (s) 7 s -89 Fura-2 Ca binding ratio (kB)

36. Conclusions : Binding = buffering & sensing Buffering reduces Ca2+ changes, Slows Ca2+ changes, Slows diffusion, Shortens local spikes of Ca2+