What is the mechanism of store-operated Ca2+ entry?

Measuring [Ca2+]ER and ICRAC in single cells

Miyawaki et al, 1999

The ER sensor for CRAC channel activation has low Ca2+ affinity

Possible candidates: Low-affinity, high-capacity (calreticulin, BiP, etc) Low-affinity, multi-EF hand (CREC family: reticulocalbin, ERC-55, etc)

References

Smani et al, 2004

Yao et al, 1999But see: Scott et al, 2003 Bakowski et al, 2003

Ma et al, 2000But see: Sugawara et al, 1997 Prakriya & Lewis, 2001

Smani et al, 2004

A recent “diffusible messenger” model for SOC activation

The fingerprint of the CRAC channel is a definitive benchmarkfor evaluating putative CRAC channel genes

Property

Mode of activation Store depletionCa-dependent inactivation Fast (ms) and slow

(sec)Pharmacology SKF-96365 2-APB La3+

Ki = 5-10 µMKi ~10 µMKi ~20 nM

Pore properties with Cao

Selectivity (PCa/PNa) ~1000 Unitary current (20 mM Ca+, -110 mV) Unitary chord conductance

-3.8 fA~ 20 fS

Pore properties without Cao: Selectivity (PCs/PNa) 0.13 Unitary current (Na+, -110 mV) Unitary chord conductance

-31 fA~ 0.2 pS

Clapham, 2003

The family of mammalian TRP channels

TRPC1: Mori et al, 2002 (DT40 knockout, inconsistent phenotype)TRPC3: Philipp et al, 2003 (CRAC mutants, rescues TCR but not TG response)TRPV6: Yue et al, 2001 (pore properties don’t match completely) Cui et al, 2002 (dom negative suppression of Icrac)

5C.C7 B6 thymocytes in B10.BR sliceTime compression: 80x, total time 60 min

Ca2+ signals in a positively selecting environment

QuickTime™ and a Video decompressor are needed to see this picture.

Positive selection

Neglect

Calcium signals during positive selection are oscillatory

Dolmetsch et al, 1998

A reductionist approach to the functions of Ca2+ dynamics in T cells:The Ca2+ clamp

Dolmetsch et al, 1998

Frequency selectivity of transcriptional activation and gene expression

PPPP

P

NFATcCalcineurin A/B

PPPP

P

Ca/Calmodulin

P

P

PPPP

P

NFAT kinase(s)

P

p65 p50

IB

P

PP

Ub

UbP

PP

IB synthesis

NFB NFAT

Calcineurin

Tomida et al, 2003

vs.

[Ca] interval

Ca2+ oscillations boost signaling efficiency

Tomida et al, 2003

Delayed phosphorylation and transport of NFAT underliesthe signaling efficiency of Ca2+ pulses

References

Review:Clapham, D.E. 2003. TRP channels as cellular sensors. Nature. 426:517-524.Prakriya, M., and R.S. Lewis. 2003. CRAC channels: activation, permeation, and the

search for a molecular identity. Cell Calcium. 33:311-321.

Original papers:Bakowski, D., R.D. Burgoyne, and A.B. Parekh. 2003. Activation of the store-operated

calcium current ICRAC can be dissociated from regulated exocytosis in RBL-1 cells.J Physiol. 553:387-393.

Cui, J., J.S. Bian, A. Kagan, and T.V. McDonald. 2002. CaT1 Contributes to the Stores-operated Calcium Current in Jurkat T-lymphocytes. J. Biol. Chem. 277:47175-47183.

Dolmetsch, R.E., K. Xu, and R.S. Lewis. 1998. Calcium oscillations increase theefficiency and specificity of gene expression. Nature. 392:933-936.

Ma, H.T., R.L. Patterson, D.B. van Rossum, L. Birnbaumer, K. Mikoshiba, and D.L. Gill.2000. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science. 287:1647-1651.

Miyawaki, A., O. Griesbeck, R. Heim, and R.Y. Tsien. 1999. Dynamic and quantitativeCa2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. 96:2135-2140.

Mori, Y., M. Wakamori, T. Miyakawa, M. Hermosura, Y. Hara, M. Nishida, K. Hirose,A. Mizushima, M. Kurosaki, E. Mori, K. Gotoh, T. Okada, A. Fleig, R. Penner,M. Iino, and T. Kurosaki. 2002. Transient receptor potential 1 regulatescapacitative Ca2+ entry and Ca2+ release from endoplasmic reticulum in Blymphocytes. J. Exp. Med. 195:673-681.

Philipp, S., B. Strauss, D. Hirnet, U. Wissenbach, L. Mery, V. Flockerzi, and M. Hoth.2003. TRPC3 mediates T-cell receptor-dependent calcium entry in human T-lymphocytes. J. Biol. Chem. 278:26629-26638.

Prakriya, M., and R.S. Lewis. 2001. Potentiation and inhibition of Ca2+ release-activatedCa2+ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently ofIP3 receptors. J. Physiol. 536:3-19.

Scott, C.C., W. Furuya, W.S. Trimble, and S. Grinstein. 2003. Activation of store-operated calcium channels: assessment of the role of snare-mediated vesiculartransport. J. Biol. Chem. 278:30534-30539.

Smani, T., S.I. Zakharov, P. Csutora, E. Leno, E.S. Trepakova, and V.M. Bolotina. 2004.A novel mechanism for the store-operated calcium influx pathway. Nat Cell Biol.6:113-120.

Sugawara, H., M. Kurosaki, M. Takata, and T. Kurosaki. 1997. Genetic evidence forinvolvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors insignal transduction through the B-cell antigen receptor. EMBO J. 16:3078-3088.

Tomida, T., K. Hirose, A. Takizawa, F. Shibasaki, and M. Iino. 2003. NFAT functions asa working memory of Ca2+ signals in decoding Ca2+ oscillation. EMBO J.22:3825-3832.

Yao, Y., A.V. Ferrer-Montiel, M. Montal, and R.Y. Tsien. 1999. Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusiblemessenger. Cell. 98:475-485.

Yue, L., J.B. Peng, M.A. Hediger, and D.E. Clapham. 2001. CaT1 manifests the poreproperties of the calcium-release-activated calcium channel. Nature. 410:705-709.